Synergistic tumor treatment with an extended pharmacokinetic IL-2 and integrin-binding-Fc fusion protein

ABSTRACT

The present invention provides a method of treating cancer with a combination of IL-2 and an integrin-binding-Fc fusion protein. The methods of the invention can be applied to a broad range of cancer types.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/US2015/044920, filed on Aug. 12, 2015,which claims priority to U.S. Provisional Patent Application No.62/036,554, filed Aug. 12, 2014. The contents of the aforementionedapplications are hereby incorporated by reference in their entireties.

GOVERNMENT FUNDING

This invention was made with Government support under contract CA174795awarded by the National Institutes of Health. The Government has certainrights in the invention.

REFERENCE TO THE SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 1, 2017, isnamed MITN_028US_SL.txt and is 163608 bytes in size.

BACKGROUND OF THE INVENTION

Interleukin-2 (IL-2) is a pleiotropic cytokine that activates andinduces the proliferation of T cells and NK cells. Although IL-2 is anFDA approved therapy, systemic IL-2 treatment has significant toxicityand therefore the response rate of patients is less than 25%. Combiningextended half-life IL-2 and an antibody against a tumor-specific antigenshows promising results for treatment. However, antibody-based therapiesoften suffer from the fact that many tumors lack known tumor-associatedantigens.

Integrins are a family of extracellular matrix adhesion receptors thatregulate a diverse array of cellular functions crucial to theinitiation, progression and metastasis of solid tumors. The importanceof integrins in tumor progression has made them an appealing target forcancer therapy and allows for the treatment of a variety of cancertypes. The integrins present on cancerous cells include α_(v)β₃,α_(v)β₅, and α₅β₁. A variety of therapeutics have been developed totarget individual integrins associated with cancer, includingantibodies, linear peptides, cyclic peptides, and peptidomimetics.However, none have utilized small, structured peptide scaffolds ortargeted more than two integrins simultaneously. Additionally, currentintegrin targeting drugs are given as a monotherapy. Novel combinationtherapies are needed to more effectively combat various cancers.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatadministration of IL-2 attached to a pharmacokinetic modifying group(hereafter referred to as “extended-pharmacokinetic (PK) IL-2”) and anintegrin-binding-Fc fusion protein provides synergistic tumor controland prolongs survival relative to monotherapy of either agent alone. Theintegrin-binding-Fc fusion protein comprises (i) an integrin-bindingpolypeptide having an integrin-binding loop and a knottin polypeptidescaffold; and (ii) an immunoglobulin Fc domain, wherein theintegrin-binding polypeptide is operably linked to the Fc domain. Animproved cancer therapy is provided that involves the combinedadministration of an effective amount of IL-2 and an integrin-binding-Fcfusion protein.

Accordingly, in one aspect, the invention provides a method for treatingcancer in a subject comprising administering to the subject an effectiveamount of interleukin (IL)-2, and an integrin-binding-Fc fusion protein,wherein the integrin-binding-Fc fusion protein comprises (i) anintegrin-binding polypeptide comprising an integrin-binding loop and anknottin polypeptide scaffold; and (ii) an immunoglobulin Fc domain,wherein the integrin-binding polypeptide is operably linked to the Fcdomain.

In one embodiment of the foregoing aspects, the IL-2 is in the form ofan extended-PK IL-2, such as an IL-2 fusion protein. In one embodiment,the fusion protein comprises an IL-2 moiety and a moiety selected fromthe group consisting of an immunoglobulin fragment, human serum albumin,and Fn3. In another embodiment, the fusion protein comprises an IL-2moiety operably linked to an immunoglobulin Fc domain. In anotherembodiment, the fusion protein comprises an IL-2 moiety operably linkedto human serum albumin. In another embodiment, the extended-PK IL-2comprises an IL-2 moiety conjugated to a non-protein polymer. In oneembodiment, the non-protein polymer is polyethylene glycol.

In one embodiment of the foregoing aspects, the integrin-binding-Fcfusion protein includes an integrin-binding polypeptide that binds to atumor associated integrin selected from the group consisting of α_(v)β₃,α_(v)β₅, and α₅β₁, or combination thereof. In one embodiment, theintegrin-binding polypeptide binds to α_(v)β₃, α_(v)β₅, and α₅β₁.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide includes an integrin-binding loop within a knottinpolypeptide scaffold. In some embodiments, the knottin polypeptidescaffold comprises at least three cysteine disulfide linkages orcrosslinked cysteine residues, and the integrin-binding loop is adjacentcysteine residues of the knottin polypeptide scaffold. In oneembodiment, the integrin-binding loop comprises an RGD peptide sequence.In another embodiment, the knottin polypeptide scaffold is derived froma knottin protein selected from the group consisting of EETI-II, AgRP,and agatoxin. In one embodiment, the knottin protein is EETI-II.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide includes an integrin-binding loop comprising an RGD peptidesequence and the knottin polypeptide scaffold is derived from EETI-II.

In one embodiment of the foregoing aspects, the knottin polypeptidescaffold is derived from EETI-II and the integrin-binding loop comprisesthe sequence, X₁X₂X₃RGDX₇X₈X₉X₁₀X₁₁, wherein each X represents any aminoacid, wherein the loop is inserted between 2 cysteine residues in theEETI-II sequence and replaces the native EETI-II sequence. In anotherembodiment, the integrin-binding loop is inserted after the firstcysteine in the native EETI-II sequence.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42or 43, wherein X₁ is selected from the group consisting of A, V, L, P,F, Y, S, H, D, and N; X₂ is selected from the group consisting of G, V,L, P, R, E, and Q; X₃ is selected from the group consisting of G, A, andP; X₇ is selected from the group consisting of W and N; X₈ is selectedfrom the group consisting of A, P, and S; X₉ is selected from the groupconsisting of P and R; X₁₀ is selected from the group consisting of A,V, L, P, S, T, and E; and X₁₁ is selected from the group consisting ofG, A, W, S, T, K, and E. In a further embodiment, theintegrin-binding-Fc fusion comprises an integrin-binding polypeptide, asset forth in SEQ ID NOs: 42 or 43, operably linked to a human IgG Fcdomain, as set forth in SEQ ID NOs: 2 or 3.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide comprises an amino acid sequence selected from the aminoacid sequences set forth in Table 1. In another embodiment, theintegrin-binding polypeptide comprises an amino acid sequence from thegroup consisting of SEQ ID NOs: 67-133. In a further embodiment, anintegrin-binding polypeptide, set forth in Table 1, is operably linkedto a human IgG1 Fc domain, set forth in SEQ ID NOs: 2 or 3.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide comprises the amino acid sequence of SEQ ID NO: 93, 94, 95or 96.

In one embodiment of the foregoing aspects, the Fc domain is a humanIgG1 Fc domain.

In one embodiment of the foregoing aspects, the integrin-bindingpolypeptide is operably linked with or without a linker to the Fcdomain. In some embodiments, the integrin-binding polypeptide is linkedto the N-terminus or C-terminus of the Fc domain without a linker. Inother embodiments, the integrin-binding polypeptide is linked to theN-terminus or C-terminus of the Fc donor with a linker such as a Gly-Serlinker.

In one embodiment of the foregoing aspects, the integrin-binding-Fcfusion protein comprises the amino acid sequence of SEQ ID NO: 48, 49,50 or 51.

In any of the foregoing aspects, the methods comprise administering IL-2and the integrin-binding-Fc fusion protein in the form of apharmaceutical composition with a pharmaceutically acceptable carrier.

In some embodiments, the integrin-binding-Fc fusion protein is in theform of a dimer.

In any of the foregoing aspects, the methods comprise administering IL-2and the integrin-binding-Fc fusion protein simultaneously orsequentially.

In any of the foregoing aspects, the methods further compriseadministering an immune checkpoint blocker, such an antibody or antibodyfragment targeting PD-1, PD-L1, CTLA-4, TIM3, LAG3, or a member of theB7 family. In one embodiment, the immune checkpoint blocker is anantibody or antibody fragment thereof targeting PD-1. In anotherembodiment, the immune checkpoint blocker is an antibody or antibodyfragment targeting CTLA4.

In certain embodiments, an antagonist of VEGF is administered in placeof an immune checkpoint blocker. In a further embodiment, the antagonistof VEGF is an antibody or antibody fragment thereof that binds VEGF, anantibody or antibody fragment thereof that binds VEGF receptor, a smallmolecule inhibitor of the VEGF receptor tyrosine kinases, a dominantnegative VEGF, or a VEGF receptor.

In one embodiment of the foregoing aspects, the methods compriseadministering an integrin-binding-Fc fusion protein and an immunecheckpoint blocker, with or without IL-2.

In any of the foregoing aspects, the cancer is selected from the groupconsisting of melanoma, leukemia, lung cancer, breast cancer, prostatecancer, ovarian cancer, colon cancer, renal cell carcinoma, pancreaticcancer, cervical cancer, and brain cancer. In some embodiments, thecancer is melanoma. In other embodiments, the cancer is brain cancer,such as medullablastoma. In other embodiments, the cancer is coloncancer.

In another aspect, the invention provides a method for inhibiting growthand/or proliferation of tumor cells in a subject comprisingadministering to the subject an effective amount of anextended-pharmacokinetic (PK) interleukin (IL)-2, and anintegrin-binding-Fc fusion protein, wherein the integrin-binding-Fcfusion protein comprises (i) an integrin-binding polypeptide comprisingan integrin-binding loop and a knottin polypeptide scaffold; and (ii) animmunoglobulin Fc domain, wherein the integrin-binding polypeptide isoperably linked to the Fc domain.

In another aspect, the invention provides a method for inhibiting growthand/or proliferation of tumor cells in a subject comprisingadministering to the subject an effective amount of anintegrin-binding-Fc fusion protein, wherein the integrin-binding-Fcfusion protein comprises (i) an integrin-binding polypeptide comprisingan integrin-binding loop and a knottin polypeptide scaffold; and (ii) animmunoglobulin Fc domain, wherein the integrin-binding polypeptide isoperably linked to the Fc domain, and an immune checkpoint blocker. In afurther embodiment, an effective amount of IL-2 is also administered.

In one embodiment of the foregoing aspect, the immune checkpoint blockeris an antibody or antibody fragment targeting PD-1. In another aspect,the immune checkpoint blocker is an antibody or antibody fragmenttargeting CTLA-4.

In another aspect, the invention provides a method for inhibiting growthand/or proliferation of tumor cells in a subject comprisingadministering to the subject an effective amount of anintegrin-binding-Fc fusion protein, wherein the integrin-binding-Fcfusion protein comprises (i) an integrin-binding polypeptide comprisingan integrin-binding loop and a knottin polypeptide scaffold; and (ii) animmunoglobulin Fc domain, wherein the integrin-binding polypeptide isoperably linked to the Fc domain, and an antagonist of VEGF. In certainembodiments, the antagonist of VEGF is an antibody or antibody fragmentthereof that binds VEGF, an antibody or antibody fragment thereof thatbinds VEGF receptor, a small molecule inhibitor of the VEGF receptortyrosine kinases, a dominant negative VEGF, or a VEGF receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings.

FIG. 1 shows the positions of the Cys-Cys disulfide linkages in thesequences of knottin proteins EETI-II (SEQ ID NO: 39), AgRP (SEQ ID NO:40) and omega agatoxin 4B (SEQ ID NO: 41). Cysteine residues can be seento be immediately flanking the RGD mimic loops, which, in the presentengineered peptides, are between the brackets. For example, in AgRP, itcan be seen that the cysteines flanking the RGD sequence will be linkedto each other, whereas in EETI they are not. The size of the graftedsequence will depend on the molecular framework structure, such thatshorter loops will be preferred in cases where they are in the frameworkadjacent to linked cysteines. Disulfide linkages for other knottinproteins are set forth in the knottin database. FIG. 1 is adapted fromBiochemistry, 40, 15520-15527 (2001) and J. Biol. Chem., 2003,278:6314-6322.

FIG. 2 is a graph comparing tumor control with TA99 and integrin bindingknottin-Fc (SEQ ID NO: 45) in subcutaneous B16F10 melanoma tumors. TA99is an antibody against TYRP-1, an antigen that is overexpressed onmelanoma cells. Tumors were established by injecting 2.5×10⁵ B16F10cells into the flanks of C57BL/6 mice. Starting on the day of tumorinoculation and every 2 days after, 80 μg knottin-Fc, 80 knottin-FcD265A, or 200 TA99 was administered. Error bars represent standard errorof the mean (SEM).

FIGS. 3A and 3B depict synergistic tumor control in established B16F10melanoma tumors. 1×10⁶ B16F10 cells were injected into the flanks ofC57BL/6 mice. 30 μg MSA/IL-2 and/or 500 μg knottin-Fc was administeredon day 6 after tumor inoculation, and every 6 days after for a total offour treatments. FIG. 3A) Tumor area graphs for each treatment. FIG. 3B)Kaplan-Meier survival plot.

FIGS. 4A and 4B depict synergistic tumor control in established MC38colon tumors. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6mice and 30 μg MSA/IL-2 and/or 500 μg knottin-Fc was administered on day6 after tumor inoculation, and every 6 days after for a total of fourtreatments. FIG. 4A) Tumor area graphs for each treatment. FIG. 4B)Kaplan-Meier survival plot.

FIGS. 5A and 5B depict synergistic tumor control in established Ag104Afibrosarcoma tumors. 1×10⁶ Ag104A cells were injected into the flanks ofC3H/HeN mice. 12.5 μg MSA/IL-2 and/or 500 μg knottin-Fc was administeredon day 6 after tumor inoculation, and every 6 days after for a total offour treatments. FIG. 5A) Tumor area graphs for each treatment. FIG. 5B)Kaplan-Meier survival plot.

FIGS. 6A and 6B depict synergistic tumor control in established B16F10melanoma tumors. 1×10⁶ B16F10 cells were injected into the flanks ofC57BL/6 mice. 30 μg MSA/IL-2 was and 500 μg knottin-Fc or knottin-FcD265A was administered on day 6 after tumor inoculation and every dayafter for a total of four treatments. FIG. 6A) Tumor area graphs foreach treatment. FIG. 6B) Kaplan-Meier survival plot.

FIGS. 7A and 7B depict synergistic tumor control in established MC38tumors. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6 miceand 30 μg MSA/IL-2 and 500 μg knottin-Fc or knottin-Fc D265A wasadministered on day 6 after tumor inoculation and every 6 days after fora total of four treatments. FIG. 7A) Tumor area graphs for eachtreatment. FIG. 7B) Kaplan-Meier survival plot.

FIGS. 8A and 8B depict synergistic tumor control with an antibody inestablished B16F10 tumors. 1×10⁶ B16F10 cells were injected into theflanks of C57BL/6 mice. 30 μg MSA/IL-2 was administered every 6 daysbeginning on day 6 after tumor inoculation for a total of 5 treatments.200 μg knottin-Fc was administered daily from days 6-30 after tumorinoculation. Antibodies against TYRP-1 (TA99) were administered at 100μg per mouse every 6 days starting on day 6 after tumor inoculation.Antibodies against PD-1 (for immune checkpoint blockade) wereadministered at 200 μg per mouse every 6 days starting on day 6 aftertumor inoculation. FIG. 8A) Tumor area graphs for each treatment. FIG.8B) Kaplan-Meier survival plot.

FIGS. 9A and 9B depict synergistic tumor control with an antibody inestablished MC38 tumors. 1×10⁶ MC38 cells were injected into the flanksof C57BL/6 mice. 30 μg MSA/IL-2, 500 μg knottin-Fc, and/or 200 μganti-PD-1 antibody was administered on day 6 after tumor inoculation andevery 6 days after for a total of four treatments. FIG. 9A) Tumor areagraphs for each treatment. FIG. 9B) Kaplan-Meier survival plot.

FIG. 10 is a line graph depicting tumor growth control comparing dosingschedules of knottin-Fc with MSA-IL-2 in the B16F10 tumor model. 30 μgMSA/IL-2 was administered on days 6, 12, 18 and 24 after tumorinoculation. 200 μg knottin-Fc was administered daily or every other daystarting 6 days after tumor inoculation. 500 μg knottin-Fc wasadministered for the weekly regiment on the same days as MSA/IL-2. Meantumor area is plotted with 5 mice per group and error bars representSEM.

FIG. 11 is a line graph depicting tumor growth control comparing dosingschedules of knottin-Fc with MSA/IL-2 in the MC38 tumor model. 30 μgMSA/IL-2 was administered on days 6, 12, 18 and 24 after tumorinoculation. 200 μg knottin-Fc was administered daily or every other daystarting 6 days after tumor inoculation. 500 μg knottin-Fc wasadministered for the weekly regiment on the same days as MSA/IL-2. Meantumor area is plotted with 5 mice per group and error bars representSEM.

FIG. 12 is a line graph depicting tumor control in subcutaneous B16F10tumors. Fc was either fused to the N-terminus (knottin-Fc) or C-terminus(Fc-knottin) to compare efficacies of fusion placement. Additionally,knottin was fused to the heavy chain of an irrelevant IgG on theC-terminus (IgG-knottin). Mice were treated with 80 μg knottin-Fc orFc-knottin or 200 μg IgG-knottin every two days starting immediatelyafter tumor inoculation.

FIG. 13 is a line graph depicting tumor control in subcutaneous MC38tumors. Fc was either fused to the N-terminus (knottin-Fc) or C-terminus(Fc-knottin) to compare efficacies of fusion placement. Additionally,knottin was fused to the heavy chain of an irrelevant IgG on theC-terminus (IgG-knottin). Mice were treated with 200 μg knottin-Fc orFc-knottin every two days starting immediately after tumor inoculation.

FIGS. 14A and 14B depict tumor control after secondary tumor challengewith MC38 tumors cells. Previously cured mice (treated with MSA/IL-2 andknottin-Fc) and age-matched naïve mice were inoculated with 1×10⁶ MC38tumor cells in the opposite flank 16-20 weeks after the initial tumorinoculation. No further treatment was administered. FIG. 14A) Tumor areagraphs for previously cured mice and age-matched naïve mice followingsecondary tumor challenge. FIG. 14B) Kaplan-Meier plot of mice subjectedto secondary tumor challenge.

FIGS. 15A and 15B depict synergistic tumor control in established B16F10melanoma tumors. 1×10⁶ B16F10 cells were injected into the flanks ofC57BL/6 mice. 30 μg MSA/IL-2, 500 μg knottin-Fc that targets 3 integrins(“2.5F_knottin-Fc”), and/or knottin-Fc that targets 2 integrins(“2.5D_knottin-Fc”), was administered on day 6 after tumor inoculation,and every 6 days after for a total of four treatments. FIG. 15A) Tumorarea graphs for each treatment. FIG. 15B) Kaplan-Meier survival plot.

FIGS. 16A and 16B depict synergistic tumor control in established MC38tumors. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6 mice.30 μg MSA/IL-2, 500 μg knottin-Fc that targets 3 integrins(“2.5F_knottin-Fc”), and/or knottin-Fc that targets 2 integrins(“2.5D_knottin-Fc”), was administered on day 6 after tumor inoculation,and every 6 days after for a total of four treatments. FIG. 16A) Tumorarea graphs for each treatment. FIG. 16B) Kaplan-Meier survival plot.

FIGS. 17A and 17B depict synergistic tumor control in established Ag104Aangiosarcoma tumors. 1×10⁶ Ag104A cells were injected into the flanks ofC3H/HeN mice. 30 μg MSA/IL-2, 500 μg knottin-Fc that targets 3 integrins(“2.5F_knottin-Fc”), and/or knottin-Fc that targets 2 integrins(“2.5D_knottin-Fc”) was administered on day 6 after tumor inoculation,and every 6 days after for a total of four treatments. FIG. 17A) Tumorarea graphs for each treatment. FIG. 17B) Kaplan-Meier survival plot.

FIGS. 18A and 18B depict the effect of immune cell depletions onsurvival of mice with established MC38 tumors treated with knottin-Fcand MSA/IL-2. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6mice. 30 μg MSA/IL-2 and 500 μg knottin-Fc was administered on day 6after tumor inoculation, and every 6 days after for a total of fourtreatments. 400 μg anti-CD8, anti-CD4, anti-NK1.1, anti-Ly6G oranti-CD19 antibodies were dosed every four days for a total of sixtreatments starting on day 4 after tumor inoculation. 300 μg anti-CSF-1Rwas dosed every two days for a total of eleven treatments starting onday 5 of tumor inoculation. 30 μg cobra venom factor (CVF) wasadministered every 6 days for a total of 4 treatments starting on day 5after tumor inoculation. FIG. 18A) Tumor area graphs for each treatment.FIG. 18B) Kaplan-Meier survival plots.

FIGS. 19A and 19B depict the effect of dendritic cell depletion onsurvival of mice with established MC38 tumors. 1×10⁶ MC38 cells wereinjected into the flanks of C57BL/6 mice or Batf3−/− mice. 30 μgMSA/IL-2 and 500 μg knottin-Fc was administered on day 6 after tumorinoculation, and every 6 days after for a total of four treatments. FIG.19A) Tumor area graphs for each treatment. FIG. 19B) Kaplan-Meiersurvival plot.

FIGS. 20A and 20B depict the role of IFNγ in the efficacy of treatmentwith MSA/IL-2 and knottin-Fc in mice with established MC38 tumors. 1×10⁶MC38 cells were injected into the flanks of C57BL/6 mice. 30 μg MSA/IL-2and 500 μg knottin-Fc was administered on day 6 after tumor inoculation,and every 6 days after for a total of four treatments. 200 μg anti-IFNγantibody was administered every two days for a total of eleventreatments starting on day 5 after tumor inoculation. FIG. 20A) Tumorarea graphs for each treatment. FIG. 20B) Kaplan-Meier survival plot.

FIGS. 21A and 21B depict synergistic tumor control in established MC38tumors. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6 mice.30 μg MSA/IL-2, 500 μg knottin-Fc, and/or 200 μg anti-VEGF antibody wasadministered on day 6 after tumor inoculation, and every 6 days afterfor a total for 4 treatments. FIG. 21A) Tumor area graphs for eachtreatment. FIG. 21B) Kaplan-Meier survival plot.

FIG. 22 shows tumor area graphs depicting synergistic tumor control inestablished MC38 tumors. 1×10⁶ MC38 cells were injected into the flanksof C57BL/6 mice. 30 μg MSA/IL-2, 500 μg knottin-Fc, and/or 200 μganti-CTLA-4 antibody was administered on day 6 after tumor inoculation,and every 6 days after for a total for 4 treatments.

FIGS. 23A and 23B depict the synergistic tumor control in establishedB16F10 tumors. 1×10⁶ B16F10 cells were injected into the flanks ofC57BL/6 mice. 30 μg MSA/IL-2, 500 μg knottin-Fc, 200 μg anti-VEGFantibody and/or 200 μg anti-CTLA-4 antibody was administered on day 6after tumor inoculation, and every 6 days after for a total for 4treatments. FIG. 23A) Tumor area graphs for each treatment. FIG. 23B).Kaplan-Meier survival plots

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant specification shall control.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups {e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid Amino acids can be referred to herein byeither their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, can be referred to by their commonlyaccepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, the presentlarger “peptide insertions,” can be made, e.g. insertion of about threeto about five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al. Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992;Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine andleucine, modifications at the second base can also be conservative. Theterm nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene. Polynucleotides used herein can be composed of anypolyribonucleotide or polydeoxribonucleotide, which can be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that can be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

As used herein, “interleukin (IL)-2,” refers to a pleiotropic cytokinethat activates and induces proliferation of T cells and natural killer(NK) cells. IL-2 signals by binding its receptor, IL-2R, which iscomprised of alpha, beta, and gamma subunits. IL-2 signaling stimulatesproliferation of antigen-activated T cells.

As used herein, the term “PK” is an acronym for “pharmacokinetic” andencompasses properties of a compound including, by way of example,absorption, distribution, metabolism, and elimination by a subject. Asused herein, an “extended-PK group” refers to a protein, peptide, ormoiety that increases the circulation half-life of a biologically activemolecule when fused to or administered together with the biologicallyactive molecule. Examples of an extended-PK group include PEG, humanserum albumin (HSA) binders (as disclosed in U.S. Publication Nos.2005/0287153 and 2007/0003549, PCT Publication Nos. WO 2009/083804 andWO 2009/133208, and SABA molecules as described in US2012/094909), humanserum albumin, Fc or Fc fragments and variants thereof, and sugars(e.g., sialic acid). Other exemplary extended-PK groups are disclosed inKontermann et al., Current Opinion in Biotechnology 2011; 22:868-876,which is herein incorporated by reference in its entirety. As usedherein, an “extended-PK IL-2” refers to an IL-2 moiety in combinationwith an extended-PK group. In one embodiment, the extended-PK IL-2 is afusion protein in which an IL-2 moiety is linked or fused to anextended-PK group. An exemplary fusion protein is an HSA/IL-2 fusion inwhich one or more IL-2 moieties are linked to HSA.

The term “extended-PK IL-2” is also intended to encompass IL-2 mutantswith mutations in one or more amino acid residues that enhance theaffinity of IL-2 for one or more of its receptors, for example, CD25. Inone embodiment, the IL-2 moiety of extended-PK IL-2 is wild-type IL-2.In another embodiment, the IL-2 moiety is a mutant IL-2 which exhibitsgreater affinity for CD25 than wild-type IL-2. When a particular type ofextended-PK group is indicated, such as HSA-IL-2, it should beunderstood that this encompasses both HSA or MSA fused to a wild-typeIL-2 moiety or HSA or MSA fused to a mutant IL-2 moiety.

In certain aspects, the extended-PK IL-2 or knottin-Fc described canemploy one or more “linker domains,” such as polypeptide linkers. Asused herein, the term “linker” or “linker domain” refers to a sequencewhich connects two or more domains (e.g., the PK moiety and IL-2) in alinear sequence. As used herein, the term “polypeptide linker” refers toa peptide or polypeptide sequence (e.g., a synthetic peptide orpolypeptide sequence) which connects two or more domains in a linearamino acid sequence of a polypeptide chain. For example, polypeptidelinkers may be used to connect an IL-2 moiety or an integrin bindingpolypeptide to an Fc domain or other PK-extender such as HSA. In someembodiments, such polypeptide linkers can provide flexibility to thepolypeptide molecule. Exemplary linkers include Gly-Ser linkers.

As used herein, the terms “linked,” “fused”, or “fusion” are usedinterchangeably. These terms refer to the joining together of two ormore elements or components or domains, by whatever means includingchemical conjugation or recombinant means. Methods of chemicalconjugation (e.g., using heterobifunctional crosslinking agents) areknown in the art.

The term “integrin” means a transmembrane heterodimeric proteinimportant for cell adhesion. Integrins comprise an α and β subunit.These proteins bind to extracellular matrix components (e.g.,fibronectin, collagen, laminin, etc.) and respond by inducing signalingcascades. Integrins bind to extracellular matrix components byrecognition of the RGD motif. Certain integrins are found on the surfaceof tumor cells and therefore make promising therapeutic targets. Incertain embodiments, the integrins being targeted are α_(v)β₃, α_(v)β₅,and α₅β₁, individually or in combination.

The term “integrin-binding polypeptide” refers to a polypeptide whichincludes an integrin-binding domain or loop within a knottin polypeptidescaffold. The integrin binding domain or loop includes at least one RGDpeptide. In certain embodiments, the RGD peptide is recognized byα_(v)β₃, α_(v)β₅, or α₅β₁. In certain embodiments the RGD peptide bindsto a combination of α_(v)β₃, α_(v)β₅, and/or α₅β₁. These specificintegrins are found on tumor cells and their vasculature and aretherefore the targets of interest.

The term “loop domain” refers to an amino acid subsequence within apeptide chain that has no ordered secondary structure, and residesgenerally on the surface of the peptide. The term “loop” is understoodin the art as referring to secondary structures that are not ordered asin the form of an alpha helix, beta sheet, etc.

The term “integrin-binding loop” refers to a primary sequence of about9-13 amino acids which is typically created ab initio throughexperimental methods such as directed molecular evolution to bind tointegrins. In certain embodiments, the integrin-binding loop includes anRGD peptide sequence, or the like, placed between amino acids which areparticular to the scaffold and the binding specificity desired. TheRGD-containing peptide or like (RYD, etc) is generally not simply takenfrom a natural binding sequence of a known protein. The integrin-bindingloop is preferably inserted within a knottin polypeptide scaffoldbetween cysteine residues, and the length of the loop adjusted foroptimal integrin-binding depending on the three-dimensional spacingbetween cysteine residues. For example, if the flanking cysteineresidues in the knottin scaffold are linked to each other, the optimalloop may be shorter than if the flanking cysteine residues are linked tocysteine residues separated in primary sequence. Otherwise, particularamino acid substitutions can be introduced to constrain a longerRGD-containing loop into an optimal conformation for high affinityintegrin binding. The knottin polypeptide scaffolds used herein maycontain certain modifications made to truncate the native knottin, or toremove a loop or unnecessary cysteine residue or disulfide bond.

Incorporation of integrin-binding sequences into a molecular (e.g.,knottin polypeptide) scaffold provides a framework for ligandpresentation that is more rigid and stable than linear or cyclic peptideloops. In addition, the conformational flexibility of small peptides insolution is high, and results in large entropic penalties upon binding.Incorporation of an integrin-binding sequence into a knottin polypeptidescaffold provides conformational constraints that are required for highaffinity integrin binding. Furthermore, the scaffold provides a platformto carry out protein engineering studies such as affinity or stabilitymaturation.

As used herein, the term “knottin protein” refers to a structural familyof small proteins, typically 25-40 amino acids, which bind to a range ofmolecular targets like proteins, sugars and lipids. Theirthree-dimensional structure is essentially defined by a peculiararrangement of three to five disulfide bonds. A characteristic knottedtopology with one disulfide bridge crossing the macro-cycle limited bythe two other intra-chain disulfide bonds, which was found in severaldifferent microproteins with the same cystine network, lent its name tothis class of biomolecules. Although their secondary structure contentis generally low, the knottins share a small triple-strandedantiparallel β-sheet, which is stabilized by the disulfide bondframework. Biochemically well-defined members of the knottin family,also called cystine knot proteins, include the trypsin inhibitor EETI-IIfrom Ecballium elaterium seeds, the neuronal N-type Ca2+ channel blockerω-conotoxin from the venom of the predatory cone snail Conus geographus,agouti-related protein (AgRP, See Millhauser et al., “Loops and Links:Structural Insights into the Remarkable Function of the Agouti-RelatedProtein,” Ann. N.Y. Acad. Sci., Jun. 1, 2003; 994(1): 27-35), the omegaagatoxin family, etc. A suitable agatoxin sequence [SEQ ID NO: 41] isgiven in U.S. Pat. No. 8,536,301, having a common inventor with thepresent application. Other agatoxin sequences suitable for use in themethods disclosed herein include, Omega-agatoxin-Aa4b (GenBank Accessionnumber P37045) and Omega-agatoxin-Aa3b (GenBank Accession numberP81744). Other knottin sequences suitable for use in the methodsdisclosed herein include, knottin [Bemisia tabaci] (GenBank Accessionnumber FJ601218.1), Omega-lycotoxin (Genbank Accession number P85079),mu-O conotoxin MrVIA=voltage-gated sodium channel blocker (GenbankAccession number AAB34917) and Momordica cochinchinensis TrypsinInhibitor I (MCoTI-I) or II (McoTI-II) (Uniprot Accession numbers P82408and P82409 respectively).

Knottin proteins have a characteristic disulfide linked structure. Thisstructure is also illustrated in Gelly et al., “The KNOTTIN website anddatabase: a new information system dedicated to the knottin scaffold,”Nucleic Acids Research, 2004, Vol. 32, Database issue D156-D159. Atriple-stranded β-sheet is present in many knottins. The spacing betweencysteine residues is important, as is the molecular topology andconformation of the integrin-binding loop.

The term “molecular scaffold” means a polymer having a predefinedthree-dimensional structure, into which an integrin-binding loop isincorporated, such as an RGD peptide sequence as described herein. Theterm “molecular scaffold” has an art-recognized meaning (in othercontexts), which is also intended here. For example, a review by Skerra,“Engineered protein scaffolds for molecular recognition,” J. Mol.Recognit. 2000; 13:167-187 describes the following scaffolds: singledomains of antibodies of the immunoglobulin superfamily, proteaseinhibitors, helix-bundle proteins, disulfide-knotted peptides andlipocalins. Guidance is given for the selection of an appropriatemolecular scaffold.

The term “knottin polypeptide scaffold” refers to a knottin proteinsuitable for use as a molecular scaffold, as described herein.Characteristics of a desirable knottin polypeptide scaffold forengineering include 1) high stability in vitro and in vivo, 2) theability to replace amino acid regions of the scaffold with othersequences without disrupting the overall fold, 3) the ability to createmultifunctional or bispecific targeting by engineering separate regionsof the molecule, and 4) a small size to allow for chemical synthesis andincorporation of non-natural amino acids if desired. Scaffolds derivedfrom human proteins are favored for therapeutic applications to reducetoxicity or immunogenicity concerns, but are not always a strictrequirement. Other scaffolds that have been used for protein designinclude fibronectin (Koide et al., 1998), lipocalin (Beste et al.,1999), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (Hufton etal., 2000), and tendamistat (McConnell and Hoess, 1995; Li et al.,2003). While these scaffolds have proved to be useful frameworks forprotein engineering, molecular scaffolds such as knottins have distinctadvantages: their small size and high stability.

As used herein, the term “EETI” means Protein Data Bank Entry (PDB)2ETI. Its entry in the Knottin database is EETI-II. It has the sequence:

(SEQ ID NO: 39) GC PRILMRCKQDSDCLAGCVCGPNGFCG.

As used herein, the term “AgRP” means PDB entry 1HYK. Its entry in theKnottin database is SwissProt AGRP_HUMAN, where the full-length sequenceof 129 amino acids may be found. It comprises the sequence beginning atamino acid 87. An additional G is added to this construct. It alsoincludes a C105A mutation described in Jackson, et al. 2002Biochemistry, 41, 7565.

(SEQ ID NO: 40) GCVRLHESCLGQQVPCCDPCATCYC RFFNAF CYCR-KLGTAMNPCSRT

The bold and underlined portion, from loop 4, is replaced by the RGDsequences described herein. Loops 1 and 3 are shown between bracketsbelow:

GC[VRLHES]CLGQQVPCC[DPCAT]CYCRFFNAFCYCR-KLGTAMNPCS RT

As used herein, “integrin-binding-Fc fusion” is used interchangeablywith “knottin-Fc” and refers to an integrin-binding polypeptide thatincludes an integrin-binding amino acid sequence within a knottinpolypeptide scaffold and is operably linked to an Fc domain. In certainembodiments, the Fc domain is fused to the N-terminus of theintegrin-binding polypeptide. In certain embodiments, the Fc domain isfused to the C-terminus of the integrin binding polypeptide. In someembodiments, the Fc domain is operably linked to the integrin-bindingpolypeptide via a linker.

As used herein, the term “Fc region” refers to the portion of a nativeimmunoglobulin formed by the respective Fc domains (or Fc moieties) ofits two heavy chains. As used herein, the term “Fc domain” refers to aportion of a single immunoglobulin (Ig) heavy chain wherein the Fcdomain does not comprise an Fv domain. As such, an Fc domain can also bereferred to as “Ig” or “IgG.” In certain embodiments, an Fc domainbegins in the hinge region just upstream of the papain cleavage site andends at the C-terminus of the antibody. Accordingly, a complete Fcdomain comprises at least a hinge domain, a CH2 domain, and a CH3domain. In certain embodiments, an Fc domain comprises at least one of:a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragmentthereof. In other embodiments, an Fc domain comprises a complete Fcdomain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In oneembodiment, an Fc domain comprises a hinge domain (or portion thereof)fused to a CH3 domain (or portion thereof). In another embodiment, an Fcdomain comprises a CH2 domain (or portion thereof) fused to a CH3 domain(or portion thereof). In another embodiment, an Fc domain consists of aCH3 domain or portion thereof. In another embodiment, an Fc domainconsists of a hinge domain (or portion thereof) and a CH3 domain (orportion thereof). In another embodiment, an Fc domain consists of a CH2domain (or portion thereof) and a CH3 domain. In another embodiment, anFc domain consists of a hinge domain (or portion thereof) and a CH2domain (or portion thereof). In one embodiment, an Fc domain lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). AnFc domain herein generally refers to a polypeptide comprising all orpart of the Fc domain of an immunoglobulin heavy-chain. This includes,but is not limited to, polypeptides comprising the entire CH1, hinge,CH2, and/or CH3 domains as well as fragments of such peptides comprisingonly, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derivedfrom an immunoglobulin of any species and/or any subtype, including, butnot limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. A human IgG1 constant region can be found at Uniprot P01857and in Table 2 (i.e., SEQ ID NO: 1) The Fc domain of human IgG1 can befound in Table 2 (i.e., SEQ ID NO: 2). The Fc domain of human IgG1 witha deletion of the upper hinge region can be found in Table 2 (i.e., SEQID NO: 3). The Fc domain encompasses native Fc and Fc variant molecules.As with Fc variants and native Fc's, the term Fc domain includesmolecules in monomeric or multimeric (e.g., dimeric) form, whetherdigested from whole antibody or produced by other means. The assignmentof amino acid residue numbers to an Fc domain is in accordance with thedefinitions of Kabat. See, e.g., Sequences of Proteins of ImmunologicalInterest (Table of Contents, Introduction and Constant Region Sequencessections), 5^(th) edition, Bethesda, Md.: NIH vol. 1:647-723 (1991);Kabat et al., “Introduction” Sequences of Proteins of ImmunologicalInterest, US Dept of Health and Human Services, NIH, 5^(th) edition,Bethesda, Md. vol. 1:xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989), each ofwhich is herein incorporated by reference for all purposes.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain exemplary embodiments, the Fc domainhas increased effector function (e.g., FcγR binding).

The Fc domains of a polypeptide of the invention may be derived fromdifferent immunoglobulin molecules. For example, an Fc domain of apolypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, an Fc domain can comprise a chimeric hinge region derived, inpart, from an IgG1 molecule and, in part, from an IgG3 molecule. Inanother example, an Fc domain can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide may have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions.

A polypeptide can comprise an amino acid sequence which is not naturallyoccurring. Such variants, in the context of IL-2 or a knottin protein,necessarily have less than 100% sequence identity or similarity with thestarting IL-2 or knottin protein. In a preferred embodiment, the variantwill have an amino acid sequence from about 75% to less than 100% aminoacid sequence identity or similarity with the amino acid sequence of thestarting polypeptide, more preferably from about 80% to less than 100%,more preferably from about 85% to less than 100%, more preferably fromabout 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%) and most preferably from about 95% to less than 100%, e.g.,over the length of the variant molecule.

In one embodiment, there is one amino acid difference between a startingpolypeptide sequence and the sequence derived therefrom. Identity orsimilarity with respect to this sequence is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical (i.e., same residue) with the starting amino acid residues,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity.

In one embodiment, a polypeptide comprising IL-2 or a variant thereof,for use in extended-PK IL-2 consists of, consists essentially of, orcomprises an amino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35. In an embodiment, apolypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to an amino acid sequence selected from SEQ IDNos: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, and 35. Inan embodiment, a polypeptide includes a contiguous amino acid sequenceat least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguousamino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, and 35. In an embodiment, a polypeptideincludes an amino acid sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous amino acids of anamino acid sequence selected from SEQ ID Nos: 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, and 35.

In an embodiment, the peptides are encoded by a nucleotide sequence.Nucleotide sequences can be useful for a number of applications,including: cloning, gene therapy, protein expression and purification,mutation introduction, DNA vaccination of a host in need thereof,antibody generation for, e.g., passive immunization, PCR, primer andprobe generation, and the like. In an embodiment, the nucleotidesequence of the invention comprises, consists of, or consistsessentially of, a nucleotide sequence of IL-2, or a variant thereof,selected from SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, and 34. In an embodiment, a nucleotide sequence includes anucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a nucleotide sequence set forth in SEQ ID Nos: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. In an embodiment, anucleotide sequence includes a contiguous nucleotide sequence at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotidesequence set forth in SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, and 34. In an embodiment, a nucleotide sequenceincludes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or500 (or any integer within these numbers) contiguous nucleotides of anucleotide sequence set forth in SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, and 34.

In one embodiment, a polypeptide comprising integrin-binding peptide ora variant thereof, consists of, consists essentially of, or comprises anamino acid sequence selected from SEQ ID Nos: 67-133. In an embodiment,a polypeptide includes an amino acid sequence at least 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to an amino acid sequence selected from SEQID Nos: 67-133. In an embodiment, a polypeptide includes a contiguousamino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto a contiguous amino acid sequence selected from SEQ ID Nos: 67-133. Inan embodiment, a polypeptide includes an amino acid sequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence selected from SEQ IDNos: 67-133.

It will also be understood by one of ordinary skill in the art that theextended-PK IL-2 or a knottin-Fc fusion used herein may be altered suchthat they vary in sequence from the naturally occurring or nativesequences from which they were derived, while retaining the desirableactivity of the native sequences. For example, nucleotide or amino acidsubstitutions leading to conservative substitutions or changes at“non-essential” amino acid residues may be made. Mutations may beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis.

The polypeptides described herein (e.g., IL-2, extended-PK IL-2, PKmoieties, knottin, Fc, knottin-Fc, and the like) may compriseconservative amino acid substitutions at one or more amino acidresidues, e.g., at essential or non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagines,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in a binding polypeptide is preferably replaced with anotheramino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members. Alternatively, in another embodiment, mutations may beintroduced randomly along all or part of a coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be incorporatedinto binding polypeptides of the invention and screened for theirability to bind to the desired target.

The “Programmed Death-1 (PD-1)” receptor refers to an immuno-inhibitoryreceptor belonging to the CD28 family. PD-1 is expressed predominantlyon previously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. AAC51773 (SEQ ID NO: 52).

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulatesT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7 (SEQ ID NO: 53).

“Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)” is a T cellsurface molecule and is a member of the immunoglobulin superfamily. Thisprotein downregulates the immune system by binding to CD80 and CD86. Theterm “CTLA-4” as used herein includes human CTLA-4 (hCTLA-4), variants,isoforms, and species homologs of hCTLA-4, and analogs having at leastone common epitope with hCTLA-4. The complete hCTLA-4 sequence can befound under GenBank Accession No. P16410 (SEQ ID NO: 54):

“Lymphocyte Activation Gene-3 (LAGS)” is an inhibitory receptorassociated with inhibition of lymphocyte activity by binding to MHCclass II molecules. This receptor enhances the function of Treg cellsand inhibits CD8+ effector T cell function. The term “LAGS” as usedherein includes human LAGS (hLAG3), variants, isoforms, and specieshomologs of hLAG3, and analogs having at least one common epitope. Thecomplete hLAG3 sequence can be found under GenBank Accession No. P18627(SEQ ID NO: 55).

“T Cell Membrane Protein-3 (TIM3)” is an inhibitory receptor involved inthe inhibition of lymphocyte activity by inhibition of T_(H)1 cellsresponses. Its ligand is galectin 9, which is upregulated in varioustypes of cancers. The term “TIM3” as used herein includes human TIM3(hTIM3), variants, isoforms, and species homologs of hTIM3, and analogshaving at least one common epitope. The complete hTIM3 sequence can befound under GenBank Accession No. Q8TDQo (SEQ ID NO: 56).

The “B7 family” refers to inhibitory ligands with undefined receptors.The B7 family encompasses B7-H3 and B7-H4, both upregulated on tumorcells and tumor infiltrating cells. The complete hB7-H3 and hB7-H4sequence can be found under GenBank Accession Nos. Q5ZPR3 and AAZ17406(SEQ ID Nos: 57 and 58) respectively.

“Vascular Endothelial Growth Factor (VEGF)” is a secreteddisulfide-linked homodimer that selectively stimulates endothelial cellsto proliferate, migrate, and produce matrix-degrading enzymes, all ofwhich are processes required for the formation of new vessels. Inaddition to being the only known endothelial cell specific mitogen, VEGFis unique among angiogenic growth factors in its ability to induce atransient increase in blood vessel permeability to macromolecules. Theterm “VEGF” or “VEGF-A” is used to refer to the 165-amino acid humanvascular endothelial cell growth factor and related 121-, 145-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by, e.g., Leung et al. Science, 246: 1306 (1989), and Houck etal. Mol. Endocrin., 5: 1806 (1991), together with the naturallyoccurring allelic and processed forms thereof. VEGF-A is part of a genefamily including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF.VEGF-A primarily binds to two high affinity receptor tyrosine kinases,VEGFR-1 (Fit-1) and VEGFR-2 (Flk-1/KDR), the latter being the majortransmitter of vascular endothelial cell mitogenic signals of VEGF-A.

As used herein, “immune checkpoint” refers to co-stimulatory andinhibitory signals that regulate the amplitude and quality of T cellreceptor recognition of an antigen. In certain embodiments, the immunecheckpoint is an inhibitory signal. In certain embodiments, theinhibitory signal is the interaction between PD-1 and PD-L1. In certainembodiments, the inhibitory signal is the interaction between CTLA-4 andCD80 or CD86 to displace CD28 binding. In certain embodiments theinhibitory signal is the interaction between LAGS and MHC class IImolecules. In certain embodiments, the inhibitory signal is theinteraction between TIM3 and galectin 9.

As used herein, “immune checkpoint blocker” refers to a molecule thatreduces, inhibits, interferes with or modulates one or more checkpointproteins. In certain embodiments, the immune checkpoint blocker preventsinhibitory signals associated with the immune checkpoint. In certainembodiments, the immune checkpoint blocker is an antibody, or fragmentthereof, that disrupts inhibitory signaling associated with the immunecheckpoint. In certain embodiments, the immune checkpoint blocker is asmall molecule that disrupts inhibitory signaling. In certainembodiments, the immune checkpoint blocker is an antibody, fragmentthereof, or antibody mimic, that prevents the interaction betweencheckpoint blocker proteins, e.g., an antibody, or fragment thereof,that prevents the interaction between PD-1 and PD-L1. In certainembodiments, the immune checkpoint blocker is an antibody, or fragmentthereof, that prevents the interaction between CTLA-4 and CD80 or CD86.In certain embodiments, the immune checkpoint blocker is an antibody, orfragment thereof, that prevents the interaction between LAGS and MHCclass II molecules. In certain embodiments the, the immune checkpointblocker is an antibody, or fragment thereof, that prevents theinteraction between TIM3 and galectin 9. The checkpoint blocker may alsobe in the form of the soluble form of the molecules (or mutationthereof) themselves, e.g., a soluble PD-L1 or PD-L1 fusion.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., cancer, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

The term “in vivo” refers to processes that occur in a living organism.

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and include but is not limited to humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As used herein, the term “gly-ser polypeptide linker” refers to apeptide that consists of glycine and serine residues. An exemplarygly-ser polypeptide linker comprises the amino acid sequenceSer(Gly₄Ser)n (SEQ ID NO: 134). In one embodiment, n=1. In oneembodiment, n=2. In another embodiment, n=3, i.e., Ser(Gly₄Ser)3 (SEQ IDNO: 135). In another embodiment, n=4, i.e., Ser(Gly₄Ser)4 (SEQ ID NO:136). In another embodiment, n=5. In yet another embodiment, n=6. Inanother embodiment, n=7. In yet another embodiment, n=8. In anotherembodiment, n=9. In yet another embodiment, n=10. Another exemplarygly-ser polypeptide linker comprises the amino acid sequence (Gly₄Ser)n(SEQ ID NO: 137). In one embodiment, n=1. In one embodiment, n=2. In apreferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6. Another exemplarygly-ser polypeptide linker comprises the amino acid sequence (Gly₃Ser)n(SEQ ID NO: 138). In one embodiment, n=1. In one embodiment, n=2. In apreferred embodiment, n=3. In another embodiment, n=4. In anotherembodiment, n=5. In yet another embodiment, n=6.

As used herein, “half-life” refers to the time taken for the serum orplasma concentration of a polypeptide to reduce by 50%, in vivo, forexample due to degradation and/or clearance or sequestration by naturalmechanisms. The extended-PK IL-2 used herein is stabilized in vivo andits half-life increased by, e.g., fusion to HSA, MSA or Fc, throughPEGylation, or by binding to serum albumin molecules (e.g., human serumalbumin) which resist degradation and/or clearance or sequestration. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may for example generally involve thesteps of suitably administering a suitable dose of the amino acidsequence or compound of the invention to a subject; collecting bloodsamples or other samples from said subject at regular intervals;determining the level or concentration of the amino acid sequence orcompound of the invention in said blood sample; and calculating, from (aplot of) the data thus obtained, the time until the level orconcentration of the amino acid sequence or compound of the inventionhas been reduced by 50% compared to the initial level upon dosing.Further details are provided in, e.g., standard handbooks, such asKenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbookfor Pharmacists and in Peters et al., Pharmacokinetic Analysis: APractical Approach (1996). Reference is also made to Gibaldi, M. et al.,Pharmacokinetics, 2^(nd) Rev. Edition, Marcel Dekker (1982).

As used herein, a “small molecule” is a molecule with a molecular weightbelow about 500 Daltons.

As used herein, “therapeutic protein” refers to any polypeptide,protein, protein variant, fusion protein and/or fragment thereof whichmay be administered to a subject as a medicament. An exemplarytherapeutic protein is an interleukin, e.g., IL-7.

As used herein, “synergy” or “synergistic effect” with regard to aneffect produced by two or more individual components refers to aphenomenon in which the total effect produced by these components, whenutilized in combination, is greater than the sum of the individualeffects of each component acting alone.

The term “sufficient amount” or “amount sufficient to” means an amountsufficient to produce a desired effect, e.g., an amount sufficient toreduce the size of a tumor.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, “combination therapy” embraces administration of eachagent or therapy in a sequential manner in a regiment that will providebeneficial effects of the combination and co-administration of theseagents or therapies in a substantially simultaneous manner Combinationtherapy also includes combinations where individual elements may beadministered at different times and/or by different routes but which actin combination to provide a beneficial effect by co-action orpharmacokinetic and pharmacodynamics effect of each agent or tumortreatment approaches of the combination therapy.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

Various aspects described herein are described in further detail in thefollowing subsections.

IL-2 and Extended-PK IL-2

Interleukin-2 (IL-2) is a cytokine that induces proliferation ofantigen-activated T cells and stimulates natural killer (NK) cells. Thebiological activity of IL-2 is mediated through a multi-subunit IL-2receptor complex (IL-2R) of three polypeptide subunits that span thecell membrane: p55 (IL-2Rα, the alpha subunit, also known as CD25 inhumans), p75 (IL-2RP, the beta subunit, also known as CD 122 in humans)and p64 (IL-2Ry, the gamma subunit, also known as CD 132 in humans). Tcell response to IL-2 depends on a variety of factors, including: (1)the concentration of IL-2; (2) the number of IL-2R molecules on the cellsurface; and (3) the number of IL-2R occupied by IL-2 (i.e., theaffinity of the binding interaction between IL-2 and IL-2R (Smith, “CellGrowth Signal Transduction is Quantal” In Receptor Activation byAntigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)).The IL-2:IL-2R complex is internalized upon ligand binding and thedifferent components undergo differential sorting. IL-2Ra is recycled tothe cell surface, while IL-2 associated with the IL-2:IL-2Rpγ complex isrouted to the lysosome and degraded. When administered as an intravenous(i.v.) bolus, IL-2 has a rapid systemic clearance (an initial clearancephase with a half-life of 12.9 minutes followed by a slower clearancephase with a half-life of 85 minutes) (Konrad et al., Cancer Res.50:2009-2017, 1990).

Thus, in some embodiments, IL-2 therapy, such as systemic IL-2, isadministered to a subject in an effective amount in combination with anintegrin-binding-Fc fusion protein, and optionally an immune checkpointblocker.

However, outcomes of systemic IL-2 administration in cancer patients arefar from ideal. While 15 to 20 percent of patients respond objectivelyto high-dose IL-2, the great majority do not, and many suffer severe,life-threatening side effects, including nausea, confusion, hypotension,and septic shock. The severe toxicity associated with IL-2 treatment islargely attributable to the activity of natural killer (NK) cells. NKcells express the intermediate-affinity receptor, IL-2Rpγ_(c), and thusare stimulated at nanomolar concentrations of IL-2, which do in factresult in patient sera during high-dose IL-2 therapy. Attempts to reduceserum concentration, and hence selectively stimulateIL-2RaPγ_(c)-bearing cells, by reducing dose and adjusting dosingregimen have been attempted, and while less toxic, such treatments werealso less efficacious. Given the toxicity issues associated with highdose IL-2 cancer therapy, numerous groups have attempted to improveanti-cancer efficacy of IL-2 by simultaneously administering therapeuticantibodies. Yet, such efforts have been largely unsuccessful, yieldingno additional or limited clinical benefit compared to IL-2 therapyalone. Accordingly, novel IL-2 therapies are needed to more effectivelycombat various cancers.

Applicants recently discovered that the ability of IL-2 to controltumors in various cancer models could be substantially increased byattaching IL-2 to a pharmacokinetic modifying group. The resultingmolecule, hereafter referred to as “extended-pharmacokinetic (PK) IL-2,”has a prolonged circulation half-life relative to free IL-2. Theprolonged circulation half-life of extended-PK IL-2 permits in vivoserum IL-2 concentrations to be maintained within a therapeutic range,leading to the enhanced activation of many types of immune cells,including T cells. Because of its favorable pharmacokinetic profile,extended-PK IL-2 can be dosed less frequently and for longer periods oftime when compared with unmodified IL-2. Extended-PK IL-2 is describedin detail in International Patent Application NO. PCT/US2013/042057,filed May 21, 2013, and claiming the benefit of priority to U.S.Provisional Patent Application No. 61/650,277, filed May 22, 2012. Theentire contents of the foregoing applications are incorporated byreference herein.

A. IL-2 and Mutants Thereof

In certain embodiments, an effective amount of human IL-2 isadministered systemically. In some embodiments, an effective amount ofan extended-PK IL-2 is administered systemically. In one embodiment, theIL-2 is a human recombinant IL-2 such as Proleukin® (aldesleukin).Proleukin® is a human recombinant interleukin-2 product produced in E.coli. Proleukin® differs from the native interleukin-2 in the followingways: a) it is not glycosylated; b) it has no N-terminal alanine; and c)it has serine substituted for cysteine at amino acid positions 125.Proleukin® exists as biologicially active, non-covalently boundmicroaggregates with an average size of 27 recombinant interleukin-2molecules. Proleukin® (aldesleukin) is administered by intravenousinfusion. In some aspects, the IL-2 portion of the extended-PK IL-2 iswild-type IL-2 (e.g., human IL-2 in its precursor form (SEQ ID NO: 33)or mature IL-2 (SEQ ID NO: 35)).

In certain embodiments, the extended-PK IL-2 is mutated such that it hasan altered affinity (e.g., a higher affinity) for the IL-2R alphareceptor compared with unmodified IL-2.

Site-directed mutagenesis can be used to isolate IL-2 mutants thatexhibit high affinity binding to CD25, i.e., IL-2Rα, as compared towild-type IL-2. Increasing the affinity of IL-2 for IL-2Rα at the cellsurface will increase receptor occupancy within a limited range of IL-2concentration, as well as raise the local concentration of IL-2 at thecell surface.

In certain embodiments, the invention features IL-2 mutants, which maybe, but are not necessarily, substantially purified and which canfunction as high affinity CD25 binders. IL-2 is a T cell growth factorthat induces proliferation of antigen-activated T cells and stimulationof NK cells. Exemplary IL-2 mutants which are high affinity bindersinclude those described in WO2013/177187A2 (herein incorporated byreference in its entirety), such as those with amino acid sequences setforth in SEQ ID Nos: 7, 23, 25, 27, 29, and 31. Further exemplary IL-2mutants with increased affinity for CD25 are disclosed in U.S. Pat. No.7,569,215, the contents of which are incorporated herein by reference.In one embodiment, the IL-2 mutant does not bind to CD25, e.g., thosewith amino acid sequences set forth in SEQ ID Nos: 9 and 11.

IL-2 mutants include an amino acid sequence that is at least 80%identical to SEQ ID NO: 33 that bind CD25. For example, an IL-2 mutantcan have at least one mutation (e.g., a deletion, addition, orsubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, or more amino acid residues) that increases the affinityfor the alpha subunit of the IL-2 receptor relative to wild-type IL-2.It should be understood that mutations identified in mouse IL-2 may bemade at corresponding residues in full length human IL-2 (nucleic acidsequence (accession: NM000586) of SEQ ID NO: 32; amino acid sequence(accession: P60568) of SEQ ID NO: 33) or human IL-2 without the signalpeptide (nucleic acid sequence of SEQ ID NO: 34; amino acid sequence ofSEQ ID NO: 35). Accordingly, in certain embodiments, the IL-2 moiety ofthe extended-PK IL-2 is human IL-2. In other embodiments, the IL-2moiety of the extended-PK IL-2 is a mutant human IL-2.

IL-2 mutants can be at least or about 50%, at least or about 65%, atleast or about 70%, at least or about 80%, at least or about 85%, atleast or about 87%, at least or about 90%, at least or about 95%, atleast or about 97%, at least or about 98%, or at least or about 99%identical in amino acid sequence to wild-type IL-2 (in its precursorform or, preferably, the mature form). The mutation can consist of achange in the number or content of amino acid residues. For example, theIL-2 mutants can have a greater or a lesser number of amino acidresidues than wild-type IL-2. Alternatively, or in addition, IL-2mutants can contain a substitution of one or more amino acid residuesthat are present in the wild-type IL-2.

By way of illustration, a polypeptide that includes an amino acidsequence that is at least 95% identical to a reference amino acidsequence of SEQ ID NO: 33 is a polypeptide that includes a sequence thatis identical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid of SEQ ID NO: 33. Forexample, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino (N-) or carboxy (C-)terminal positions of the reference amino acid sequence or anywherebetween those terminal positions, interspersed either individually amongresidues in the reference sequence or in one or more contiguous groupswithin the reference sequence.

The substituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. These mutations can be atamino acid residues that contact IL-2Rα.

In general, the polypeptides used in the practice of the instantinvention will be synthetic, or produced by expression of a recombinantnucleic acid molecule. In the event the polypeptide is an extended-PKIL-2 (e.g., a fusion protein containing at least IL-2 and a heterologouspolypeptide, such as a hexa-histidine tag or hemagglutinin tag or an Fcregion or human serum albumin), it can be encoded by a hybrid nucleicacid molecule containing one sequence that encodes IL-2 and a secondsequence that encodes all or part of the heterologous polypeptide.

The techniques that are required to make IL-2 mutants are routine in theart, and can be performed without resort to undue experimentation by oneof ordinary skill in the art. For example, a mutation that consists of asubstitution of one or more of the amino acid residues in IL-2 can becreated using a PCR-assisted mutagenesis technique (e.g., as known inthe art and/or described herein for the creation of IL-2 mutants).Mutations that consist of deletions or additions of amino acid residuesto an IL-2 polypeptide can also be made with standard recombinanttechniques. In the event of a deletion or addition, the nucleic acidmolecule encoding IL-2 is simply digested with an appropriaterestriction endonuclease. The resulting fragment can either be expresseddirectly or manipulated further by, for example, ligating it to a secondfragment. The ligation may be facilitated if the two ends of the nucleicacid molecules contain complementary nucleotides that overlap oneanother, but blunt-ended fragments can also be ligated. PCR-generatednucleic acids can also be used to generate various mutant sequences.

In addition to generating IL-2 mutants via expression of nucleic acidmolecules that have been altered by recombinant molecular biologicaltechniques, IL-2 mutants can be chemically synthesized. Chemicallysynthesized polypeptides are routinely generated by those of skill inthe art.

As noted above, IL-2 can also be prepared as fusion or chimericpolypeptides that include IL-2 and a heterologous polypeptide (i.e., apolypeptide that is not IL-2). The heterologous polypeptide can increasethe circulating half-life of the chimeric polypeptide in vivo, and may,therefore, further enhance the properties of IL-2. As discussed infurther detail infra, the polypeptide that increases the circulatinghalf-life may be serum albumin, such as human or mouse serum albumin.

In other embodiments, the chimeric polypeptide can include IL-2 and apolypeptide that functions as an antigenic tag, such as a FLAG sequence.FLAG sequences are recognized by biotinylated, highly specific,anti-FLAG antibodies, as described herein (see also Blanar et al.,Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA89:8145, 1992). In certain embodiments, the chimeric polypeptide furthercomprises a C-terminal c-myc epitope tag.

Chimeric polypeptides can be constructed using no more than conventionalmolecular biological techniques, which are well within the ability ofthose of ordinary skill in the art to perform.

a. Nucleic Acid Molecules Encoding IL-2

IL-2, either alone or as a part of a chimeric polypeptide, such as thosedescribed herein, can be obtained by expression of a nucleic acidmolecule. Thus, nucleic acid molecules encoding polypeptides containingIL-2 or an IL-2 mutant are considered within the scope of the invention,such as those with nucleic acid sequences set forth in SEQ ID Nos: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34. Just as IL-2mutants can be described in terms of their identity with wild-type IL-2,the nucleic acid molecules encoding them will necessarily have a certainidentity with those that encode wild-type IL-2. For example, the nucleicacid molecule encoding an IL-2 mutant can be at least 50%, at least 65%,preferably at least 75%, more preferably at least 85%, and mostpreferably at least 95% (e.g., 99%) identical to the nucleic acidencoding full length wild-type IL-2 (e.g., SEQ ID NO: 32 or wild-typeIL-2 without the signal peptide (e.g., SEQ ID NO: 34).

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. These nucleic acid molecules can consist of RNA or DNA(for example, genomic DNA, cDNA, or synthetic DNA, such as that producedby phosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

The nucleic acid molecules are not limited to sequences that encodepolypeptides; some or all of the non-coding sequences that lie upstreamor downstream from a coding sequence (e.g., the coding sequence of IL-2)can also be included. Those of ordinary skill in the art of molecularbiology are familiar with routine procedures for isolating nucleic acidmolecules. They can, for example, be generated by treatment of genomicDNA with restriction endonucleases, or by performance of the polymerasechain reaction (PCR). In the event the nucleic acid molecule is aribonucleic acid (RNA), molecules can be produced, for example, by invitro transcription.

The isolated nucleic acid molecules can include fragments not found assuch in the natural state. Thus, the invention encompasses use ofrecombinant molecules, such as those in which a nucleic acid sequence(for example, a sequence encoding an IL-2 mutant) is incorporated into avector (e.g., a plasmid or viral vector) or into the genome of aheterologous cell (or the genome of a homologous cell, at a positionother than the natural chromosomal location).

As described above, IL-2 mutants of the invention may exist as a part ofa chimeric polypeptide. In addition to, or in place of, the heterologouspolypeptides described above, a nucleic acid molecule of the inventioncan contain sequences encoding a “marker” or “reporter.” Examples ofmarker or reporter genes include β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (neon, G4189, dihydrofolate reductase (DHFR),hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), lacz(encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures associatedwith the practice of the invention, skilled artisans will be aware ofadditional useful reagents, for example, of additional sequences thatcan serve the function of a marker or reporter.

The nucleic acid molecules of the invention can be obtained byintroducing a mutation into IL-2-encoding DNA obtained from anybiological cell, such as the cell of a mammal. Thus, the nucleic acidsused herein (and the polypeptides they encode) can be those of a mouse,rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, orcat. Typically, the nucleic acid molecules will be those of a human.

B. Extended-PK Groups

As described supra, IL-2 or mutant IL-2 is fused to an extended-PKgroup, which increases circulation half-life. Non-limiting examples ofextended-PK groups are described infra. It should be understood thatother PK groups that increase the circulation half-life of IL-2, orvariants thereof, are also applicable to extended-PK IL-2.

In certain embodiments, the serum half-life of extended-PK IL-2 isincreased relative to IL-2 alone (i.e., IL-2 not fused to an extended-PKgroup). In certain embodiments, the serum half-life of extended-PK IL-2is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or1000% longer relative to the serum half-life of IL-2 alone. In otherembodiments, the serum half-life of the extended-PK IL-2 is at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold,20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or50-fold greater than the serum half-life of IL-2 alone. In certainembodiments, the serum half-life of the extended-PK IL-2 is at least 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours.

a. Serum Albumin and Serum Albumin Binding Proteins

In certain embodiments, the extended-PK group is a serum albumin, orfragments thereof. Methods of fusing serum albumin to proteins aredisclosed in, e.g., US2010/0144599, US2007/0048282, and US2011/0020345,which are herein incorporated by reference in their entirety. In certainembodiments, the extended-PK group is HSA, or variants or fragmentsthereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO2011/124718, WO 2013/075066, and WO 2011/0514789.

In certain embodiments, the extended-PK group is a serum albumin bindingprotein such as those described in US2005/0287153, US2007/0003549,US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, andWO2009/133208, which are herein incorporated by reference in theirentirety.

b. PEGylation

In certain embodiments, an extended-PK IL-2 used herein includes apolyethylene glycol (PEG) domain. PEGylation is well known in the art toconfer increased circulation half-life to proteins. Methods ofPEGylation are well known and disclosed in, e.g., U.S. Pat. Nos.7,610,156, 7,847,062, all of which are hereby incorporated by reference.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X-0(CH₂CH₂O)_(n-1)CH₂CH₂OH,where n is 20 to 2300 and X is H or a terminal modification, e.g., aC₁₋₄ alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”).PEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462, both of which are hereby incorporated by reference. Oneform of PEGs includes two PEG side-chains (PEG2) linked via the primaryamino groups of a lysine (Monfardini et al., Bioconjugate Chem 1995;6:62-9).

In one embodiment, pegylated IL-2 is produced by site-directedpegylation, particularly by conjugation of PEG to a cysteine moiety atthe N- or C-terminus. A PEG moiety may also be attached by otherchemistry, including by conjugation to amines.

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., JBC 1977; 252:3571 and JBC 1977; 252:3582, and Harriset. al., in: Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications; (J. M. Harris ed.) Plenum Press: New York,1992; Chap. 21 and 22).

A variety of molecular mass forms of PEG can be selected, e.g., fromabout 1,000 Daltons (Da) to 100,000 Da (n is 20 to 2300), forconjugating to IL-2. The number of repeating units “n” in the PEG isapproximated for the molecular mass described in Daltons. It ispreferred that the combined molecular mass of PEG on an activated linkeris suitable for pharmaceutical use. Thus, in one embodiment, themolecular mass of the PEG molecules does not exceed 100,000 Da. Forexample, if three PEG molecules are attached to a linker, where each PEGmolecule has the same molecular mass of 12,000 Da (each n is about 270),then the total molecular mass of PEG on the linker is about 36,000 Da(total n is about 820). The molecular masses of the PEG attached to thelinker can also be different, e.g., of three molecules on a linker twoPEG molecules can be 5,000 Da each (each n is about 110) and one PEGmolecule can be 12,000 Da (n is about 270).

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated IL-2 will be used therapeutically, thedesired dosage, circulation time, resistance to proteolysis,immunogenicity, and other considerations. For a discussion of PEG andits use to enhance the properties of proteins, see N. V. Katre, AdvancedDrug Delivery Reviews 1993; 10:91-114.

In one embodiment of the invention, PEG molecules may be activated toreact with amino groups on IL-2 such as with lysines (Bencham C. O. etal., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl.Biochem., 11, 141 (1985); Zalipsky, S. et al., Polymeric Drugs and DrugDelivery Systems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky,S. et al., Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al.,Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

In one embodiment, carbonate esters of PEG are used to form the PEG-IL-2conjugates. N,N′-disuccinimidylcarbonate (DSC) may be used in thereaction with PEG to form active mixed PEG-succinimidyl carbonate thatmay be subsequently reacted with a nucleophilic group of a linker or anamino group of IL-2 (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In asimilar type of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of IL-2 can be performed according to the methods of thestate of the art, for example by reaction of IL-2 with electrophilicallyactive PEGs (Shearwater Corp., USA, www.shearwatercorp.com). PreferredPEG reagents are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA),butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C, et al.,Bioconjugate Chem. 6 (1995) 62-69).

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson IL-2 (Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991);Morpurgo et al., Biocon. Chem., 7, 363-368 (1996); Goodson et al.,Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat. Nos.6,610,281 and 5,766,897 describe exemplary reactive PEG species that maybe coupled to sulfhydryl groups.

In certain embodiments where PEG molecules are conjugated to cysteineresidues on IL-2 the cysteine residues are native to IL-2 whereas inother embodiments, one or more cysteine residues are engineered intoIL-2. Mutations may be introduced into the coding sequence of IL-2 togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein.

In another embodiment, pegylated IL-2 comprise one or more PEG moleculescovalently attached to a linker.

In one embodiment, IL-2 is pegylated at the C-terminus. In a specificembodiment, a protein is pegylated at the C-terminus by the introductionof C-terminal azido-methionine and the subsequent conjugation of amethyl-PEG-triarylphosphine compound via the Staudinger reaction. ThisC-terminal conjugation method is described in Cazalis et al., C-TerminalSite-Specific PEGylation of a Truncated Thrombomodulin Mutant withRetention of Full Bioactivity, Bioconjug Chem. 2004; 15(5): 1005-1009.

Monopegylation of IL-2 can also be achieved according to the generalmethods described in WO 94/01451. WO 94/01451 describes a method forpreparing a recombinant polypeptide with a modified terminal amino acidalpha-carbon reactive group. The steps of the method involve forming therecombinant polypeptide and protecting it with one or more biologicallyadded protecting groups at the N-terminal alpha-amine and C-terminalalpha-carboxyl. The polypeptide can then be reacted with chemicalprotecting agents to selectively protect reactive side chain groups andthereby prevent side chain groups from being modified. The polypeptideis then cleaved with a cleavage reagent specific for the biologicalprotecting group to form an unprotected terminal amino acid alpha-carbonreactive group. The unprotected terminal amino acid alpha-carbonreactive group is modified with a chemical modifying agent. The sidechain protected terminally modified single copy polypeptide is thendeprotected at the side chain groups to form a terminally modifiedrecombinant single copy polypeptide. The number and sequence of steps inthe method can be varied to achieve selective modification at the N-and/or C-terminal amino acid of the polypeptide.

The ratio of IL-2 to activated PEG in the conjugation reaction can befrom about 1:0.5 to 1:50, between from about 1:1 to 1:30, or from about1:5 to 1:15. Various aqueous buffers can be used to catalyze thecovalent addition of PEG to IL-2, or variants thereof. In oneembodiment, the pH of a buffer used is from about 7.0 to 9.0. In anotherembodiment, the pH is in a slightly basic range, e.g., from about 7.5 to8.5. Buffers having a pKa close to neutral pH range may be used, e.g.,phosphate buffer.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated IL-2, such as size exclusion (e.g. gelfiltration) and ion exchange chromatography. Products may also beseparated using SDS-PAGE. Products that may be separated include mono-,di-, tri-poly- and un-pegylated IL-2 as well as free PEG. The percentageof mono-PEG conjugates can be controlled by pooling broader fractionsaround the elution peak to increase the percentage of mono-PEG in thecomposition.

In one embodiment, PEGylated IL-2 of the invention contains one, two ormore PEG moieties. In one embodiment, the PEG moiety(ies) are bound toan amino acid residue which is on the surface of the protein and/or awayfrom the surface that contacts CD25. In one embodiment, the combined ortotal molecular mass of PEG in PEG-IL-2 is from about 3,000 Da to 60,000Da, optionally from about 10,000 Da to 36,000 Da. In one embodiment, PEGin pegylated IL-2 is a substantially linear, straight-chain PEG.

In one embodiment, PEGylated IL-2 of the invention will preferablyretain at least 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% of thebiological activity associated with the unmodified protein. In oneembodiment, biological activity refers to the ability to bind CD25. Theserum clearance rate of PEG-modified IL-2 may be decreased by about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative to theclearance rate of the unmodified IL-2. PEG-modified IL-2 may have acirculation half-life which is enhanced relative to the half-life ofunmodified IL-2. The half-life of PEG-IL-2, or variants thereof, may beenhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by 1000%relative to the half-life of unmodified IL-2. In certain embodiments,the protein half-life is determined in vitro, such as in a bufferedsaline solution or in serum. In other embodiments, the protein half-lifeis an in vivo circulation half-life, such as the half-life of theprotein in the serum or other bodily fluid of an animal.

c. Other Extended-PK Groups

In certain embodiments, the extended-PK group is transferrin, asdisclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579, which are hereinincorporated by reference in their entirety.

In certain embodiments, the extended-PK group is a serum immunoglobulinbinding protein such as those disclosed in US2007/0178082, which isherein incorporated by reference in its entirety.

In certain embodiments, the extended-PK group is a fibronectin(Fn)-based scaffold domain protein that binds to serum albumin, such asthose disclosed in US2012/0094909, which is herein incorporated byreference in its entirety. Methods of making fibronectin-based scaffolddomain proteins are also disclosed in US2012/0094909. A non-limitingexample of a Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3protein that binds to human serum albumin.

d. Fc Domains

In certain embodiments, an extended-PK IL-2 includes an Fc domain, asdescribed in WO2013177187. The Fc domain does not contain a variableregion that binds to antigen. Fc domains useful for producing theextended-PK IL-2 described herein may be obtained from a number ofdifferent sources. In certain embodiments, an Fc domain of theextended-PK IL-2 is derived from a human immunoglobulin. In a certainembodiment, the Fc domain is from a human IgG1 constant region (SEQ IDNO: 1). The Fc domain of human IgG1 is set forth in SEQ ID NO: 2. Incertain embodiments, the Fc domain of human IgG1 does not have the upperhinge region (SEQ ID NO: 3). It is understood, however, that the Fcdomain may be derived from an immunoglobulin of another mammalianspecies, including for example, a rodent (e.g. a mouse, rat, rabbit,guinea pig) or non-human primate (e g chimpanzee, macaque) species.Moreover, the Fc domain or portion thereof may be derived from anyimmunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and anyimmunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

In some aspects, an extended-PK IL-2 includes a mutant Fc domain. Insome aspects, an extended-PK IL-2 includes a mutant, IgG1 Fc domain. Insome aspects, a mutant Fc domain comprises one or more mutations in thehinge, CH2, and/or CH3 domains. In some aspects, a mutant Fc domainincludes a D265A mutation.

In one embodiment, the extended-PK IL-2 of the invention lacks one ormore constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In certain embodiments, the extended-PKIL-2 of the invention will lack an entire CH2 domain. In certainembodiments, the extended-PK IL-2 of the invention comprise CH2domain-deleted Fc regions derived from a vector (e.g., from IDECPharmaceuticals, San Diego) encoding an IgG1 human constant regiondomain (see, e.g., WO02/060955A2 and WO02/096948A2). This exemplaryvector is engineered to delete the CH2 domain and provide a syntheticvector expressing a domain-deleted IgG1 constant region. It will benoted that these exemplary constructs are preferably engineered to fusea binding CH3 domain directly to a hinge region of the respective Fcdomain.

Integrin-Binding-Fc Fusion Proteins

Integrins are a family of extracellular matrix adhesion receptors thatregulate a diverse array of cellular functions crucial to theinitiation, progression and metastasis of solid tumors. The importanceof integrins in tumor progression has made them an appealing target forcancer therapy and allows for the treatment of a variety of cancertypes. The integrins present on cancerous cells include α_(v)β₃,α_(v)β₅, and α₅β₁.

Knottin proteins are small compact peptides that have high thermal andproteolytic stability and are tolerant to mutagenesis, making them goodmolecular scaffolds. These peptides contain at least 3 disulfide bondsthat form a “knot” core. They also contain several loops exposed to thesurface, allowing these loops to bind targets. These loops can beengineered to bind specific targets with high affinity, making them auseful tool for therapy.

The present invention involves the use of a knottin polypeptide scaffoldengineered with an RGD sequence capable of binding integrins, fused toan Fc donor, which confers a therapeutic benefit (also referred to as“knottin-Fc”). As described supra, Fc fragments have been added toproteins and/or therapeutics to extend half-life. In the context ofknottin-Fc as used herein, the effector function of Fc contributes tothe treatment of a variety of cancers when used in conjunction withsystemic IL-2, such as extended-PK IL-2. In certain embodiments, aknottin-Fc that binds two integrins simultaneously is used (2.5D, SEQ IDNO: 93 or 95). In certain embodiments, a knottin-Fc that binds threeintegrins simultaneously, reflected in Table 1, is used (2.5F, SEQ IDNO: 94 or 96).

A. Methods of Engineering Knottin Polypeptide Scaffolds

Knottin polypeptide scaffolds are used to insert an integrin-bindingsequence, preferably in the form of a loop, to confer specific integrinbinding. Integrin-binding is preferably engineered into a knottinpolypeptide scaffold by inserting an integrin-binding peptide sequence,such as an RGD peptide. In some embodiments, insertion of anintegrin-binding peptide sequence results in replacement of portion ofthe native knottin protein. For example, in one embodiment an RGDpeptide sequence is inserted into a native solvent exposed loop byreplacing all or a portion of the loop with an RGD-containing peptidesequence (e.g., 5-12 amino acid sequence) that has been selected forbinding to one or more integrins. The solvent-exposed loop (i.e., on thesurface) will generally be anchored by disulfide-linked cysteineresidues in the native knottin protein sequence. The integrin-bindingreplacement amino acid sequence can be obtained by randomizing codons inthe loop portion, expressing the engineered peptide, and selecting themutants with the highest binding to the predetermined ligand. Thisselection step may be repeated several times, taking the tightestbinding proteins from the previous step and re-randomizing the loops.

Integrin-binding polypeptides may be modified in a number of ways. Forexample, the polypeptide may be further cross-linked internally, or maybe cross-linked to each other, or the RGD loops may be grafted ontoother cross linked molecular scaffolds. There are a number ofcommercially available crosslinking reagents for preparing protein orpeptide bioconjugates. Many of these crosslinkers allow dimeric homo- orheteroconjugation of biological molecules through free amine orsulfhydryl groups in protein side chains. More recently, othercrosslinking methods involving coupling through carbohydrate groups withhydrazide moieties have been developed. These reagents have offeredconvenient, facile, crosslinking strategies for researchers with littleor no chemistry experience in preparing bioconjugates.

The EETI-II knottin protein (SEQ ID NO: 39) contains a disulfide knottedtopology and possesses multiple solvent-exposed loops that are amenableto mutagenesis. Preferred embodiments use EETI-II as the molecularscaffold.

Another example of a knottin protein which can be used as a molecularscaffold is AgRP or agatoxin. The amino acid sequences of AgRP (SEQ IDNO: 40) and agatoxin (SEQ ID NO: 41) differ but their structure isidentical. Exemplary AgRP knottins are found in Table 1.

Additional AgRP engineered knottins can be made as described in theabove-referenced US 2009/0257952 to Cochran et al. (the contents ofwhich are incorporated herein by reference). AgRP knottin fusions can beprepared using AgRP loops 1, 2 and 3, as well as loop 4 as exemplifiedabove.

The present polypeptides may be produced by recombinant DNA or may besynthesized in solid phase using a peptide synthesizer, which has beendone for the peptides of all three scaffolds described herein. They mayfurther be capped at their N-termini by reaction with fluoresceinisothiocyanate (FITC) or other labels, and, still further, may besynthesized with amino acid residues selected for additionalcrosslinking reactions. TentaGel S RAM Fmoc resin (Advanced ChemTech)may be used to give a C-terminal amide upon cleavage. B-alanine is usedas the N-terminal amino acid to prevent thiazolidone formation andrelease of fluorescein during peptide deprotection (Hermanson, 1996).Peptides are cleaved from the resin and side-chains are deprotected with8% trifluoroacetic acid, 2% triisopropylsilane, 5% dithiothreitol, andthe final product is recovered by ether precipitation. Peptides arepurified by reverse phase HPLC using an acetonitrile gradient in 0.1%trifluoroacetic acid and a C4 or C18 column (Vydac) and verified usingmatrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF) or electrospray ionization-mass spectrometry(ESI-MS).

When the present peptides are produced by recombinant DNA, expressionvectors encoding the selected peptide are transformed into a suitablehost. The host should be selected to ensure proper peptide folding anddisulfide bond formation as described above. Certain peptides, such asEETI-II, can fold properly when expressed in prokaryotic hosts such asbacteria.

Dimeric, trimeric, and tetrameric complexes of the present peptides canbe formed through genetic engineering of the above sequences or byreaction of the synthetic crosslinkers with engineered peptides carryingan introduced cysteine residue, for example on the C-terminus of thepeptide. These oligomeric peptide complexes can be purified by gelfiltration. Oligomers of the present peptides can be prepared bypreparing vectors encoding multiple peptide sequences end-to-end. Also,multimers may be prepared by complexing the peptides, such as, e.g.,described in U.S. Pat. No. 6,265,539. There, an active HIV peptide isprepared in multimer form by altering the amino-terminal residue of thepeptide so that it is peptide-bonded to a spacer peptide that containsan amino-terminal lysyl residue and one to about five amino acidresidues such as glycyl residues to form a composite polypeptide.Alternatively, each peptide is synthesized to contain a cysteine (Cys)residue at each of its amino- and carboxy-termini. The resultingdi-cysteine-terminated (di-Cys) peptide is then oxidized to polymerizethe di-Cys peptide monomers into a polymer or cyclic peptide multimer.Multimers may also be prepared by solid phase peptide synthesisutilizing a lysine core matrix. The present peptides may also beprepared as nanoparticles. See, “Multivalent Effects of RGD PeptidesObtained by Nanoparticle Display,” Montet, et al., J. Med. Chem.; 2006;49 (20) pp 6087-6093. EETI dimerization may be carried out with thepresent EETI-II peptides according to the EETI-II dimerization paper:“Grafting of thrombopoietin-mimetic peptides into cystine knotminiproteins yields high-affinity thrombopoietin antagonist andagonists,” Krause, et al., FEBS Journal; 2006; 274 pp 86-95. This isfurther described in PCT application No. PCT/US2013/065610, hereinincorporated by reference.

Synergistic sites on fibronectin and other adhesion proteins have beenidentified for enhanced integrin binding (Ruoslahti, 1996; Koivunen etal., 1994; Aota et al., 1994; Healy et al., 1995). The ability toincorporate different integrin-specific motifs into one soluble moleculewould have an important impact on therapeutic development. Crosslinkerswith heterofunctional specificity may be used for creatingintegrin-binding proteins with synergistic binding effects. In addition,these same crosslinkers could easily be used to create bispecifictargeting molecules, or as vehicles for delivery of radionuclides ortoxic agents for therapeutic applications.

B. Integrin-Binding Peptides

The integrin-binding polypeptides for use in Fc fusions include anintegrin-binding loop (e.g., RGD peptide sequence) and a knottinpolypeptide scaffold. Such integrin-binding polypeptides are describedin U.S. Pat. No. 8,536,301, the contents of which are incorporatedherein by reference. As described in U.S. Pat. No. 8,536,301,integrin-binding polypeptides may be varied in the non-RGD residues to acertain degree without affecting binding specificity and potency. Forexample, if three of the eleven residues were varied, one would haveabout 70% identity to 2.5D. Table 1 shows exemplary integrin-bindingpolypeptides within the scope of the invention, and their specificknottin polypeptide scaffold (e.g., EETI-II or AgRP). Preferredintegrin-binding polypeptides for use in Fc fusions are peptides 2.5Dand 2.5F.

In certain embodiments, the integrin-binding polypeptide binds toα_(v)β₃, α_(v)β₅, or α₅β₁ separately.

In certain embodiments, the integrin-binding polypeptide binds toα_(v)β₃ and α_(v)β₅ simultaneously.

In certain embodiments, the integrin-binding polypeptide binds toα_(v)β₃, α_(v)β₅, and α₅β₁ simultaneously.

In certain embodiments, the integrin-binding loop is within anengineered EETI-II scaffold. In certain embodiments, the lysine inposition 15 of the EETI-II scaffold is replaced with a serine. Incertain embodiments, the integrin-binding polypeptide comprises theamino acid sequence set forth in SEQ ID NO: 42 or 43, wherein X₁ isselected from the group consisting of A, V, L, P, F, Y, S, H, D, and N;X₂ is selected from the group consisting of G, V, L, P, R, E, and Q; X₃is selected from the group consisting of G, A, and P; X₇ is selectedfrom the group consisting of W and N; X₈ is selected from the groupconsisting of A, P, and S; X₉ is selected from the group consisting of Pand R; X₁₀ is selected from the group consisting of A, V, L, P, S, T,and E; and X₁₁ is selected from the group consisting of G, A, W, S, T,K, and E. In a further embodiment, the integrin-binding-Fc fusioncomprises an integrin-binding polypeptide, as set forth in SEQ ID Nos:42 or 43, operably linked to a human IgG Fc domain, as set forth in SEQID Nos: 2 or 3.

In certain embodiments, the integrin-binding loop is within anengineered AgRP or agatoxin scaffold.

In certain embodiments, the integrin-binding polypeptide is 2.5D and2.5F, disclosed in Table 1. Any of the integrin-binding polypeptides inTable 1 can be used in Fc fusion as described herein.

TABLE 1 Integrin Binding Knottin Sequences SEQ ID PeptideSequence (RGD motif is underlined with flanking NO Identifier Scaffoldresidues) 67 1.4A EETI-II GC AEPRGDMPWTW CKQDSDCLAGCVCGPNGFCG 68 1.4BEETI-II GC VGGRGDWSPKW CKQDSDCPAGCVCGPNGFCG 69 1.4C EETI-II GC AELRGDRSYPE  CKQDSDCLAGCVCGPNGFCG 70 1.4E EETI-II GC  RLPRGDVPRPH CKQDSDCQAGCVCGPNGFCG 71 1.4H EETI-II GC  YPLRGDNPYAA CKQDSDCRAGCVCGPNGFCG 72 1.5B EETI-II GC  TIGRGDWAPSE CKQDSDCLAGCVCGPNGFCG 73 1.5F EETI-II GC  HPPRGDNPPVT CKQDSDCLAGCVCGPNGFCG 74 2.3A EETI-II GC  PEPRGDNPPPS CKQDSDCRAGCVCGPNGFCG 75 2.3B EETI-II GC  LPPRGDNPPPS CKQDSDCQAGCVCGPNGFCG 76 2.3C EETI-II GC HLGRGDWAPVG CKQDSDCPAGCVCGPNGFCG77 2.3D EETI-II GC  NVGRGDWAPSE CKQDSDCPAGCVCGPNGFCG 78 2.3E EETI-II GC FPGRGDWAPSS CKQDSDCRAGCVCGPNGFCG 79 2.3F EETI-II GC  PLPRGDNPPTE CKQDSDCQAGCVCGPNGFCG 80 2.3G EETI-II GC  SEARGDNPRLS CKQDSDCRAGCVCGPNGFCG 81 2.3H EETI-II GC LLGRGDWAPEA CKQDSDCRAGCVCPNGFCG82 2.3I EETI-II GC HVGRGDWAPLK CKQDSDCQAGCVCGPNGFCG 83 2.3J EETI-II GC VRGRGDWAPPS CKQDSDCPAGCVCGPNGFCG 84 2.4A EETI-II GC LGGRGDWAPPACKQDSDCRAGCVCGPNGFCG 85 2.4C EETI-II GC  FVGRGDWAPLTCKQDSDCQAGCVCGPNGFCG 86 2.4D EETI-II GC  PVGRGDWSPASCKQDSDCRAGCVCGPNGFCG 87 2.4E EETI-II GC  PRPRGDNPPLT CKQDSDCLAGCVCGPNGFCG 88 2.4F EETI-II GC  YQGRGDWSPSSCKQDSDCPAGCVCGPNGFCG 89 2.4G EETI-II GC  APGRGDWAPSECKQDSDCQAGCVCGPNGFCG 90 2.4J EETI-II GC  VQGRGDWSPPSCKQDSDCPAGCVCGPNGFCG 91 2.5A EETI-II GC  HVGRGDWAPEECKQDSDCQAGCVCGPNGFCG 92 2.5C EETI-II GC  DGGRGDWAPPACKQDSDCRAGCVCGPNGFCG 93 2.5D EETI-II GC  PQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG 94 2.5F EETI-II GC  PRPRGDNPPLT CKQDSDCLAGCVCGPNGFCG 95 2.5D K15S EETI-II GC PQGRGDWAPTSCSQDSDCLAGCVCGPNGFCG Mutant 96 2.5F K15S EETI-II GC PRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG Mutant 97 2.5H EETI-II GC PQGRGDWAPEWCKQDSDCPAGCVCGPNGFCG 98 2.5J EETI-II GC PRGRGDWSPPA CKQDSDCQAGCVCGPNGFCG99 3A AgRp GCVRLHESCLGQQVPCCDPAATCYC VVRGDWRKR CYCR 100 3B AgRpGCVRLHESCLGQQVPCCDPAATCYC  EERGDMLEK CYCR 101 3C AgRpGCVRLHESCLGQQVPCCDPAATCYC  ETRGDGKEK CYCR 102 3D AgRpGCVRLHESCLGQQVPCCDPAATCYC QWRGDGDVK CYCR 103 3E AgRpGCVRLHESCLGQQVPCCDPAATCYC SRRGDMRER CYCR 104 3F AgRpGCVRLHESCLGQQVPCCDPAATCYC QYRGDGMKH CYCR 105 3G AgRpGCVRLHESCLGQQVPCCDPAATCYC  TGRGDTKVL CYCR 106 3H AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDMKRR CYCR 107 3I AgRpGCVRLHESCLGQQVPCCDPAATCYC  TGRGDVRMN CYCR 108 3J AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGMSK CYCR 109 4A AgRpGCVRLHESCLGQQVPCCDPAATCYC RGRGDMRRE CYCR 110 4B AgRpGCVRLHESCLGQQVPCCDPAATCYC  EGRGDVKVN CYCR 111 4C AgRpGCVRLHESCLGQQVPCCDPAATCYC VGRGDEKMS CYCR 112 4D AgRpGCVRLHESCLGQQVPCCDPAATCYC VSRGDMRKR CYCR 113 4E AgRpGCVRLHESCLGQQVPCCDPAATCYC  ERRGDSVKK CYCR 114 4F AgRpGCVRLHESCLGQQVPCCDPAATCYC  EGRGDTRRR CYCR 115 4G AgRpGCVRLHESCLGQQVPCCDPAATCYC  EGRGDVVRR CYCR 116 4H AgRpGCVRLHESCLGQQVPCCDPAATCYC KGRGDNKRK CYCR 117 4I AgRpGCVRLHESCLGQQVPCCDPAXTCYC  KGRGDVRRV CYCR 118 4J AgRpGCVRLHESCLGQQVPCCDPAATCYC  VGRGDNKVK CYCR 119 5A AgRpGCVRLHESCLGQQVPCCDPAATCYC  VGRGDNRLK CYCR 120 5B AgRpGCVRLHESCLGQQVPCCDPAATCYC VERGDGMKK CYCR 121 5C AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDMRRR CYCR 122 5D AgRpGCVRLHESCLGQQVPCCDPAATCYC QGRGDGDVK CYCR 123 5E AgRpGCVRLHESCLGQQVPCCDPAATCYC  SGRGDNDLV CYCR 124 5F AgRpGCVRLHESCLGQQVPCCDPAATCYC  VERGDGMIR CYCR 125 5G AgRpGCVRLHESCLGQQVPCCDPAATCYC  SGRGDNDLV CYCR 126 5H AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDMKMK CYCR 127 5I AgRpGCVRLHESCLGQQVPCCDPAATCYC  IGRGDVRRR CYCR 128 5J AgRpGCVRLHESCLGQQVPCCDPAATCYC  EERGDGRKK CYCR 129 6B AgRpGCVRLHESCLGQQVPCCDPAATCYC EGRGDRDMK CYCR 130 6C AgRpGCVRLHESCLGQQVPCCDPAATCYC  TGRGDEKLR CYCR 131 6E AgRpGCVRLHESCLGQQVPCCDPAATCYC  VERGDGNRR CYCR 132 6F AgRpGCVRLHESCLGQQVPCCDPAATCYC  ESRGDVVRK CYCR 133 7C AgRpGCVRLHESCLGQQVPCCDPAATCYCYGRGDNDLRCYCR

The present polypeptides target α_(v)β₃, α_(v)β₅, and in some cases α₅β₁integrin receptors. They do not bind to other integrins tested, such asα_(iib)β₃, where there was little to no affinity. Thus, these engineeredintegrin-binding polypeptides have broad diagnostic and therapeuticapplications in a variety of human cancers that specifically overexpressthe above named integrins. As described below, these polypeptides bindwith high affinity to both detergent-solubilized and tumor cell surfaceintegrin receptors.

The α_(v)β₃ (and α_(v)β₅) integrins are also highly expressed on manytumor cells including osteosarcomas, neuroblastomas, carcinomas of thelung, breast, prostate, and bladder, glioblastomas, and invasivemelanomas The α_(v)β₃ integrin has been shown to be expressed on tumorcells and/or the vasculature of breast, ovarian, prostate, and coloncarcinomas, but not on normal adult tissues or blood vessels. Also, theα₅β₁ integrin has been shown to be expressed on tumor cells and/or thevasculature of breast, ovarian, prostate, and colon carcinomas, but noton normal adult tissue or blood vessels. The present, small,conformationally-constrained polypeptides (about 33 amino acids) are soconstrained by intramolecular bonds. For example, EETI-II has threedisulfide linkages. This will make it more stable in vivo. Thesepeptides target α_(v) integrins alone, or both α_(v) and α₅β₁ integrins.Until now, it is believed that the development of a single agent thatcan bind α_(v)β₃, α_(v)β₅, and α₅β₁ integrins with high affinity andspecificity has not been achieved. Since all three of these integrinsare expressed on tumors and are involved in mediating angiogenesis andmetastasis, a broad spectrum targeting agent (i.e., α_(v)β₃, α_(v)β₅,and as α₅β₁) will likely be more effective for diagnostic andtherapeutic applications.

The present engineered knottin-Fc fusions have several advantages overpreviously identified integrin-targeting compounds. They possess acompact, disulfide-bonded core that confers proteolytic resistance andexceptional in vivo stability.

Our studies indicate the half-life of integrin-binding-Fc fusion proteinin mouse serum to be greater than 90 hours. Their larger size (^(˜)3-4kDa) and enhanced affinity compared to RGD-based cyclic peptides conferenhanced pharmacokinetics and biodistribution for molecular imaging andtherapeutic applications. These knottin-Fc proteins are small enough toallow for chemical synthesis and site-specific conjugation of imagingprobes, radioisotopes, or chemotherapeutic agents. Furthermore, they caneasily be chemically modified to further improve in vivo properties ifnecessary.

C. Knottin-Fc Fusion

The knottin-Fc fusions described herein and in U.S. Patent ApplicationNo. 2014/0073518, herein incorporated by reference in its entirety,combine an engineered integrin-binding polypeptide (within a knottinscaffold) and an Fc domain or antibody like construct capable of bindingFcγR and inducing ADCC.

The Fc portion of an antibody is formed by the two carboxy terminaldomains of the two heavy chains that make up an immunoglobin molecule.The IgG molecule contains 2 heavy chains (^(˜)50 kDa each) and 2 lightchains (^(˜)25 kDa each). The general structure of all antibodies isvery similar, a small region at the tip of the protein is extremelyvariable, allowing millions of antibodies with slightly different tipstructures to exist. This region is known as the hypervariable region(Fab). The other fragment contains no antigen-binding activity but wasoriginally observed to crystallize readily, and for this reason wasnamed the Fc fragment, for Fragment crystallizable. This fragmentcorresponds to the paired CH₂ and CH₃ domains and is the part of theantibody molecule that interacts with effector molecules and cells. Thefunctional differences between heavy-chain isotypes lie mainly in the Fcfragment. The hinge region that links the Fc and Fab portions of theantibody molecule is in reality a flexible tether, allowing independentmovement of the two Fab arms, rather than a rigid hinge. This has beendemonstrated by electron microscopy of antibodies bound to haptens. Thusthe present fusion proteins can be made to contain two knottin peptides,one on each arm of the antibody fragment.

The Fc portion varies between antibody classes (and subclasses) but isidentical within that class. The C-terminal end of the heavy chain formsthe Fc region. The Fc region plays an important role as a receptorbinding portion. The Fc portion of antibodies will bind to Fc receptorsin two different ways. For example, after IgG and IgM bind to a pathogenby their Fab portion their Fc portions can bind to receptors onphagocytic cells (like macrophages) inducing phagocytosis.

The present knottin-Fc fusions can be implemented such that the Fcportion is used to provide dual binding capability, and/or for half-lifeextension, for improving expression levels, etc. The Fc fragment in theknottin-Fc can be, for example, from murine IgG2a or human IgG1. Linkerscan be optionally used to connect the knottin to the Fc portion.Preferably, the linkers do not affect the binding affinity of theknottin-Fc to integrins or Fc receptors. A variety of Fc domain genesequences (e.g., mouse and human constant region gene sequences) areavailable in the form of publicly accessible deposits.

a. Fc-Domains

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g., hinge, CH2, and/or CH3 sequences, or portionsthereof) can be derived from these sequences using art recognizedtechniques. The genetic material obtained using any of the foregoingmethods may then be altered or synthesized to obtain polypeptides usedherein. It will further be appreciated that alleles, variants andmutations of constant region DNA sequences are suitable for use in themethods disclosed herein.

Knottin-Fc suitable for use in the methods disclosed herein may compriseone or more Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fcdomains). In one embodiment, the Fc domains may be of different types.In one embodiment, at least one Fc domain present in a knottin-Fccomprises a hinge domain or portion thereof. In another embodiment, aknottin-Fc comprises at least one Fc domain which comprises at least oneCH2 domain or portion thereof. In another embodiment, a knottin-Fccomprises at least one Fc domain which comprises at least one CH3 domainor portion thereof. In another embodiment, a knottin-Fc comprises atleast one Fc domain which comprises at least one CH4 domain or portionthereof. In another embodiment, a knottin-Fc comprises at least one Fcdomain which comprises at least one hinge domain or portion thereof andat least one CH2 domain or portion thereof (e.g, in the hinge-CH2orientation). In another embodiment, a knottin-Fc comprises at least oneFc domain which comprises at least one CH2 domain or portion thereof andat least one CH3 domain or portion thereof (e.g, in the CH2-CH3orientation). In another embodiment, a knottin-Fc comprises at least oneFc domain comprising at least one hinge domain or portion thereof, atleast one CH2 domain or portion thereof, and least one CH3 domain orportion thereof, for example in the orientation hinge-CH2-CH3,hinge-CH3-CH2, or CH2-CH3-hinge.

In certain embodiments, a knottin-Fc comprises at least one complete Fcregion derived from one or more immunoglobulin heavy chains (e.g., an Fcdomain including hinge, CH2, and CH3 domains, although these need not bederived from the same antibody). In other embodiments a knottin-Fccomprises at least two complete Fc domains derived from one or moreimmunoglobulin heavy chains. In certain embodiments, the complete Fcdomain is derived from a human IgG immunoglobulin heavy chain (e.g.,human IgG1).

In another embodiment, a knottin-Fc comprises at least one Fc domaincomprising a complete CH3 domain. In another embodiment, a knottin-Fccomprises at least one Fc domain comprising a complete CH2 domain. Inanother embodiment, a knottin-Fc comprises at least one Fc domaincomprising at least a CH3 domain, and at least one of a hinge region,and a CH2 domain. In one embodiment, a knottin-Fc comprises at least oneFc domain comprising a hinge and a CH3 domain. In another embodiment, aknottin-Fc comprises at least one Fc domain comprising a hinge, a CH2,and a CH3 domain. In certain embodiments, the Fc domain is derived froma human IgG immunoglobulin heavy chain (e.g., human IgG1). In certainembodiments, a human IgG1 Fc domain is used with a hinge regionmutation, substitution, or deletion to remove or substitute one or morehinge region cysteine residues.

The constant region domains or portions thereof making up an Fc domainof a knottin-Fc may be derived from different immunoglobulin molecules.For example, a polypeptide used in the invention may comprise a CH2domain or portion thereof derived from an IgG1 molecule and a CH3 regionor portion thereof derived from an IgG3 molecule. In another example, aknottin-Fc can comprise an Fc domain comprising a hinge domain derived,in part, from an IgG1 molecule and, in part, from an IgG3 molecule. Asset forth herein, it will be understood by one of ordinary skill in theart that an Fc domain may be altered such that it varies in amino acidsequence from a naturally occurring antibody molecule.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, a peptidespacer may be placed between a hinge region and a CH2 domain and/orbetween a CH2 and a CH3 domain. For example, compatible constructs couldbe expressed wherein the CH2 domain has been deleted and the remainingCH3 domain (synthetic or unsynthetic) is joined to the hinge region witha 1-20, 1-10, or 1-5 amino acid peptide spacer. Such a peptide spacermay be added, for instance, to ensure that the regulatory elements ofthe constant region domain remain free and accessible or that the hingeregion remains flexible. Preferably, any linker peptide compatible withthe instant invention will be relatively non-immunogenic and not preventproper folding of the Fc.

b. Changes to Fc Amino Acids

In certain embodiments, an Fc domain is altered or modified, e.g., byamino acid mutation (e.g., addition, deletion, or substitution). As usedherein, the term “Fc domain variant” refers to an Fc domain having atleast one amino acid modification, such as an amino acid substitution,as compared to the wild-type Fc from which the Fc domain is derived. Forexample, wherein the Fc domain is derived from a human IgG1 antibody, avariant comprises at least one amino acid mutation (e.g., substitution)as compared to a wild type amino acid at the corresponding position ofthe human IgG1 Fc region.

In certain embodiments, the hinge region of human IgG1 Fc domain isaltered by an amino acid substitution or deletion to mutate or removeone or more of three hinge region cysteine residues (located at residues220, 226, and 229 by EU numbering). In some aspects, the upper hingeregion is deleted to remove a cysteine that pairs with the light chain.For example, amino acids “EPKSC” in the upper hinge region are deleted,as set forth in SEQ ID NO: 3. In other aspects, one or more of threehinge region cysteines is mutated (e.g., to serine). In certainembodiments, cysteine 220 is mutated to serine.

In certain embodiments, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

In certain embodiments, a knottin-Fc fusion comprises an Fc variantcomprising more than one amino acid substitution. The knottin-Fc fusionused in the methods described herein may comprise, for example, 2, 3, 4,5, 6, 7, 8, 9, 10 or more amino acid substitutions. Preferably, theamino acid substitutions are spatially positioned from each other by aninterval of at least 1 amino acid position or more, for example, atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. Morepreferably, the engineered amino acids are spatially positioned apartfrom each other by an interval of at least 5, 10, 15, 20, or 25 aminoacid positions or more.

In certain embodiments, a knottin-Fc fusion comprises an amino acidsubstitution to an Fc domain which alters the antigen-independenteffector functions of the polypeptide, in particular the circulatinghalf-life of the polypeptide.

In one embodiment, the knottin-Fc exhibits enhanced binding to anactivating FcγR (e.g. Fcγl, Fcγla, or FcγRIIIa). Exemplary amino acidsubstitutions which altered FcR or complement binding activity aredisclosed in International PCT Publication No. WO 2005/063815 which isincorporated by reference herein. In certain embodiments the Fc regioncontains at least one of the following mutations: S239D, S239E, L261A,H268D, S298A, A330H, A330L, I332D, I332E, I332Q, K334V, A378F, A378K,A378W, A378Y, H435S, or H435G. In certain embodiments, the Fc regioncontains at least one of the following mutations: S239D, S239E, I332D orI332E or H268D. In certain embodiments, the Fc region contains at leastone of the following mutations: I332D or I332E or H268D.

The knottin-Fc used herein may also comprise an amino acid substitutionwhich alters the glycosylation of the knottin-Fc. For example, the Fcdomain of the knottin-Fc may comprise an Fc domain having a mutationleading to reduced glycosylation (e.g., N- or O-linked glycosylation) ormay comprise an altered glycoform of the wild-type Fc domain (e.g., alow fucose or fucose-free glycan). In another embodiment, the knottin-Fchas an amino acid substitution near or within a glycosylation motif, forexample, an N-linked glycosylation motif that contains the amino acidsequence NXT or NXS. Exemplary amino acid substitutions which reduce oralter glycosylation are disclosed in WO05/018572 and US2007/0111281,which are incorporated by reference herein. In other embodiments, theknottin-Fc used herein comprises at least one Fc domain havingengineered cysteine residue or analog thereof which is located at thesolvent-exposed surface. In certain embodiments, the knottin-Fc usedherein comprises an Fc domain comprising at least one engineered freecysteine residue or analog thereof that is substantially free ofdisulfide bonding with a second cysteine residue. Any of the aboveengineered cysteine residues or analogs thereof may subsequently beconjugated to a functional domain using art-recognized techniques (e.g.,conjugated with a thiol-reactive heterobifunctional linker).

In one embodiment, the knottin-Fc used herein may comprise a geneticallyfused Fc domain having two or more of its constituent Fc domainsindependently selected from the Fc domains described herein. In oneembodiment, the Fc domains are the same. In another embodiment, at leasttwo of the Fc domains are different. For example, the Fc domains of theknottin-Fc used herein comprise the same number of amino acid residuesor they may differ in length by one or more amino acid residues (e.g.,by about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acidresidues), about 10 residues, about 15 residues, about 20 residues,about 30 residues, about 40 residues, or about 50 residues). In yetother embodiments, the Fc domains of the knottin-Fc used herein maydiffer in sequence at one or more amino acid positions. For example, atleast two of the Fc domains may differ at about 5 amino acid positions(e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about15 positions, about 20 positions, about 30 positions, about 40positions, or about 50 positions).

Immune Checkpoint Blocker

In certain embodiments, immune checkpoint blockers are used incombination with other therapeutic agents described herein (e.g.,extended-PK IL-2 and integrin-binding-Fc fusion protein). T cellactivation and effector functions are balanced by co-stimulatory andinhibitory signals, referred to as “immune checkpoints.” Inhibitoryligands and receptors that regulate T cell effector functions areoverexpressed on tumor cells. Subsequently, agonists of co-stimulatoryreceptors or antagonists of inhibitory signals, result in theamplification of antigen-specific T cell responses. In contrast totherapeutic antibodies which target tumor cells directly, immunecheckpoint blockers enhance endogenous anti-tumor activity. In certainembodiments, the immune checkpoint blocker suitable for use in themethods disclosed herein, is an antagonist of inhibitory signals, e.g.,an antibody which targets, for example, PD-1, PD-L1, CTLA-4, LAGS,B7-H3, B7-H4, and TIM3. These ligands and receptors are reviewed inPardoll, D., Nature. 12: 252-264, 2012.

Disclosed herein are methods for treating a subject afflicted withdiseases such as cancer, which methods comprise administering to thesubject a composition comprising a therapeutically effective amount of amolecule which blocks the immune checkpoint, and an integrin-binding-Fcfusion protein. In certain embodiments, the methods for treating asubject afflicted with diseases such as cancer, which methods compriseadministering to the subject a composition comprising a therapeuticallyeffective amount of a molecule which blocks the immune checkpoint, anintegrin-binding-Fc fusion protein, and IL-2 (e.g., extended-PK IL-2).In certain embodiments, the immune checkpoint blocker is an antibody oran antigen-binding portion thereof, that disrupts or inhibits signalingfrom an inhibitory immunoregulator. In certain embodiments, the immunecheckpoint blocker is a small molecule that disrupts or inhibitssignaling from an inhibitory immunoregulator.

In certain embodiments, the inhibitory immunoregulator (immunecheckpoint blocker) is a component of the PD-1/PD-L1 signaling pathway.Accordingly, certain embodiments provide methods for immunotherapy of asubject afflicted with cancer, which methods comprise administering tothe subject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that disrupts the interaction betweenthe PD-1 receptor and its ligand, PD-L1. Antibodies known in the artwhich bind to PD-1 and disrupt the interaction between the PD-1 and itsligand, PD-L1, and stimulates an anti-tumor immune response, aresuitable for use in the methods disclosed herein. In certainembodiments, the antibody or antigen-binding portion thereof bindsspecifically to PD-1. For example, antibodies that target PD-1 include,e.g., nivolumab (BMS-936558, Bristol-Myers Squibb) and pembrolizumab(lambrolizumab, MK03475, Merck). Other suitable antibodies for use inthe methods disclosed herein are anti-PD-1 antibodies disclosed in U.S.Pat. No. 8,008,449, herein incorporated by reference. In certainembodiments, the antibody or antigen-binding portion thereof bindsspecifically to PD-L1 and inhibits its interaction with PD-1, therebyincreasing immune activity. Antibodies known in the art which bind toPD-L1 and disrupt the interaction between the PD-1 and PD-L1, andstimulates an anti-tumor immune response, are suitable for use in themethods disclosed herein. For example, antibodies that target PD-L1 andare in clinical trials, include BMS-936559 (Bristol-Myers Squibb) andMPDL3280A (Genetech). Other suitable antibodies that target PD-L1 aredisclosed in U.S. Pat. No. 7,943,743, herein incorporated by reference.It will be understood by one of ordinary skill that any antibody whichbinds to PD-1 or PD-L1, disrupts the PD-1/PD-L1 interaction, andstimulates an anti-tumor immune response, are suitable for use in themethods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe CTLA-4 signaling pathway. Accordingly, certain embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets CTLA-4 and disrupts its interaction with CD80 and CD86.Exemplary antibodies that target CTLA-4 include ipilimumab (MDX-010,MDX-101, Bristol-Myers Squibb), which is FDA approved, and tremelimumab(ticilimumab, CP-675, 206, Pfizer), currently undergoing human trials.Other suitable antibodies that target CTLA-4 are disclosed in WO2012/120125, U.S. Pat. Nos. 6,984,720, 6,682,7368, and U.S. PatentApplications 2002/0039581, 2002/0086014, and 2005/0201994, hereinincorporated by reference. It will be understood by one of ordinaryskill that any antibody which binds to CTLA-4, disrupts its interactionwith CD80 and CD86, and stimulates an anti-tumor immune response, aresuitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe LAG3 (lymphocyte activation gene 3) signaling pathway. Accordingly,certain embodiments provide methods for immunotherapy of a subjectafflicted with cancer, which methods comprise administering to thesubject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets LAG3 and disrupts itsinteraction with MHC class II molecules. An exemplary antibody thattargets LAG3 is IMP321 (Immutep), currently undergoing human trials.Other suitable antibodies that target LAG3 are disclosed in U.S. PatentApplication 2011/0150892, herein incorporated by reference. It will beunderstood by one of ordinary skill that any antibody which binds toLAG3, disrupts its interaction with MHC class II molecules, andstimulates an anti-tumor immune response, are suitable for use in themethods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe B7 family signaling pathway. In certain embodiments, the B7 familymembers are B7-H3 and B7-H4. Accordingly, certain embodiments providemethods for immunotherapy of a subject afflicted with cancer, whichmethods comprise administering to the subject a therapeuticallyeffective amount of an antibody or an antigen-binding portion thereofthat targets B7-H3 or -H4. The B7 family does not have any definedreceptors but these ligands are upregulated on tumor cells ortumor-infiltrating cells. Preclinical mouse models have shown thatblockade of these ligands can enhance anti-tumor immunity. An exemplaryantibody that targets B7-H3 is MGA271 (Macrogenics), currentlyundergoing human trials. Other suitable antibodies that target B7 familymembers are disclosed in U.S. Patent Application 2013/0149236, hereinincorporated by reference. It will be understood by one of ordinaryskill that any antibody which binds to B7-H3 or H4, and stimulates ananti-tumor immune response, are suitable for use in the methodsdisclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe TIM3 (T cell membrane protein 3) signaling pathway. Accordingly,certain embodiments provide methods for immunotherapy of a subjectafflicted with cancer, which methods comprise administering to thesubject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets TIM3 and disrupts itsinteraction with galectin 9. Suitable antibodies that target TIM3 aredisclosed in U.S. Patent Application 2013/0022623, herein incorporatedby reference. It will be understood by one of ordinary skill that anyantibody which binds to TIM3, disrupts its interaction with galectin 9,and stimulates an anti-tumor immune response, are suitable for use inthe methods disclosed herein.

It should be understood that antibodies targeting immune checkpointssuitable for use in the methods disclosed herein are not limited tothose described supra. Moreover, it will be understood by one ofordinary skill in the art that other immune checkpoint targets can alsobe targeted by antagonists or antibodies in the methods describedherein, provided that the targeting results in the stimulation of ananti-tumor immune response as reflected in, e.g., an increase in T cellproliferation, enhanced T cell activation, and/or increased cytokineproduction (e.g., IFN-γ, IL-2).

Alternatives to Immune Checkpoint Blockers

In certain embodiments, an antagonist of vascular endothelial growthfactor (VEGF) is used in place of an immune checkpoint blocker. VEGF hasrecently been demonstrated to play a role in immune suppression (Liang,W.-C. et al. J. Biol. Chem. (2006) Vol 281: 951-961; Voron, T. et al.Front Oncol (2014) Vol. 4: Article 70; Terme, M. et al., Clin DevImmunol (2012) Vol. 2012: Article ID 492920; Kandalaft, E. et al., CurrTop Microbiol Immunol (2011) Vol 344: 129-48), therefore blocking itsactivity would enhance the immune response, similar to that of an immunecheckpoint blocker. A “VEGF antagonist” refers to a molecule capable ofneutralizing, blocking, inhibiting, abrogating, reducing or interferingwith VEGF activities including its binding to one or more VEGFreceptors. Non-limiting examples of VEGF antagonists include anti-VEGFantibodies and antigen-binding fragments thereof, receptor molecules andderivatives which bind specifically to VEGF thereby sequestering itsbinding to one or more receptors (e.g., a VEGF receptor), anti-VEGFreceptor antibodies, VEGF receptor antagonists such as small moleculeinhibitors of the VEGFR tyrosine kinases, or a dominant negative VEGF.

In certain embodiments, the VEGF antagonist is an antibody. An“anti-VEGF antibody” is an antibody that binds to VEGF with sufficientaffinity and specificity. Non-limiting examples of anti-VEGF antibodiesare described in U.S. Pat. Nos. 6,884,879, 7,060,269, 6,582,959,6,703,030, 6,054,297, US Patent Application Nos. 2006009360,20050186208, 20030206899, 20030190317, 20030203409, 20050112126, and PCTPublication Nos. WO 98/45332, 96/30046, 94/10202, 05/044853, 13/181452.The contents of these patents and patent applications are hereinincorporated by reference. In certain embodiments the VEGF antibody isbevacizumab (Avastin® Genentech/Roche) or ranibizumab (Lucentis®Genentech/Roche).

In certain embodiments, the VEGF antagonist binds to the VEGF receptor.VEGF receptors, or fragments thereof, that specifically bind to VEGF canbe used to bind to and sequester the VEGF protein, thereby preventing itfrom activating downstream signaling. In certain embodiments, the VEGFreceptor, or VEGF binding fragment thereof, is a soluble VEGF receptor,such as sFlt-1. The soluble form of the receptor exerts an inhibitoryeffect on the biological activity of VEGF by binding to VEGF, therebypreventing it from binding to its natural receptors present on thesurface of target cells. Non-limiting examples of VEGF antagonists whichbind the VEGF receptor are disclosed in PCT Application Nos. 97/44453,05/000895 and U.S. Patent Application No. 20140057851. In certainembodiments the VEGF antagonist is a polypeptide with a bifunctionalsingle-chain antagonistic human VEGF variant comprising a modified VEGFwherein the modified VEGF comprises a loop with an integrin-recognitionRGD sequence, as described in U.S. Pat. No. 8,741,839, hereinincorporated by reference.

Linkers

In certain embodiments, the extended-PK group is optionally fused toIL-2 via a linker. In certain embodiments, an integrin-bindingpolypeptide is fused to an Fc fragment via a linker. Suitable linkersare well known in the art, such as those disclosed in, e.g.,US2010/0210511 US2010/0179094, and US2012/0094909, which are hereinincorporated by reference in its entirety. Exemplary linkers includegly-ser polypeptide linkers, glycine-proline polypeptide linkers, andproline-alanine polypeptide linkers. In a certain embodiment, the linkeris a gly-ser polypeptide linker, i.e., a peptide that consists ofglycine and serine residues.

Exemplary gly-ser polypeptide linkers comprise the amino acid sequenceSer(Gly₄Ser)n (SEQ ID NO: 134). In one embodiment, n=1. In oneembodiment, n=2. In another embodiment, n=3, i.e., Ser(Gly₄Ser)3 (SEQ IDNO: 135). In another embodiment, n=4, i.e., Ser(Gly₄Ser)4 (SEQ ID NO:136). In another embodiment, n=5. In yet another embodiment, n=6. Inanother embodiment, n=7. In yet another embodiment, n=8. In anotherembodiment, n=9. In yet another embodiment, n=10. Another exemplarygly-ser polypeptide linker comprises (Gly₄Ser)n (SEQ ID NO: 137). In oneembodiment, n=1. In one embodiment, n=2. In a certain embodiment, n=3.In another embodiment, n=4. In another embodiment, n=5. In yet anotherembodiment, n=6. Another exemplary gly-ser polypeptide linker comprises(Gly₃Ser)n (SEQ ID NO: 138). In one embodiment, n=1. In one embodiment,n=2. In a certain embodiment, n=3. In another embodiment, n=4. Inanother embodiment, n=5. In yet another embodiment, n=6.

Other Therapeutic Agents

The integrin-binding-Fc fusion protein suitable for use in the methodsdisclosed herein, can be used in conjunction with one or moretherapeutic agents. In one embodiment, the therapeutic agent is atherapeutic antibody. In another embodiment, the therapeutic agent is atherapeutic protein. In another embodiment, the therapeutic agent is asmall molecule. In another embodiment, the therapeutic agent is anantigen. In another embodiment, the therapeutic agent is a population ofcells.

Engineered Fusion Molecules

Also provided herein are engineered molecules that comprise two or moreof IL-2, and an antibody (e.g., a therapeutic antibody, an immunecheckpoint blocker, or an antibody that antagonizes VEGF) or antibodyfragment described herein. Such engineered molecules can effectivelyreduce the number of components to be administered to a subject (e.g., acancer patient) in the methods described herein. In some embodiments,the antibody or antibody fragment serves as the scaffold for conjugationwith other components (e.g., IL-2).

Accordingly, in certain embodiments, the engineered molecule comprisesIL-2 and an antibody or antibody fragment. In a particular embodiment,the antibody for use in the engineered protein is a bispecific antibody,wherein one component is a therapeutic antibody and the other componentis an antibody that binds to an immune checkpoint blocker or an antibodythat antagonizes VEGF activity. Methods for generating bispecificantibodies are known in the art.

Accordingly, in certain embodiments, the engineered molecule comprisesIL-2 and a bispecific antibody which binds to a therapeutic target andan immune checkpoint blocker or an antibody that antagonizes VEGF.

In certain embodiments, the IL-2 component for use in the engineeredprotein is an IL-2 lacking a pharmacokinetic moiety (i.e., anon-extended-PK IL-2). In other embodiments, the IL-2 comprises apharmacokinetic moiety (an extended-PK IL-2).

In certain embodiments, the components of the engineered molecule areconjugated to the antibody or bispecific antibody with or without alinker. Suitable linkers for conjugation are described herein andextensively described in the art.

Regions to which polypeptide-based components (e.g., IL-2) of theengineered molecule can be fused, with or without a linker, to theantibody are generally known in the art, and include, for example, theC-terminus of the antibody heavy chain, and the C-terminus of theantibody light chain.

In certain embodiments, components of the engineered molecule do notinterfere with the function of the other components. By way of example,when the engineered protein comprises a therapeutic antibody and IL-2,the IL-2 will be fused to the therapeutic antibody in a manner such thatthe antibody retains its antigen-binding function, and IL-2 retains theability to interact with its receptor. The methods described herein,e.g., in the Examples, can be used to determine whether components ofthe engineered protein retain their respective functions.

Methods of Making Polypeptides

In some aspects, the polypeptides described herein (e.g., IL-2, such asextended-PK IL-2, and knottin-Fc) are made in transformed host cellsusing recombinant DNA techniques. To do so, a recombinant DNA moleculecoding for the peptide is prepared. Methods of preparing such DNAmolecules are well known in the art. For instance, sequences coding forthe peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The methods of making polypeptides also include a vector capable ofexpressing the peptides in an appropriate host. The vector comprises theDNA molecule that codes for the peptides operatively linked toappropriate expression control sequences. Methods of affecting thisoperative linking, either before or after the DNA molecule is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal nuclease domains,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. colisp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3^(rd) ed.) 2: 105-253; and Erickson et al. (1976), TheProteins (3^(rd) ed.) 2: 257-527. Solid phase synthesis is the preferredtechnique of making individual peptides since it is the mostcost-effective method of making small peptides. Compounds that containderivatized peptides or which contain non-peptide groups may besynthesized by well-known organic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

Expression of Polypeptides

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto extended-PK IL-2 and knottin-Fc mutants, expression vectorscontaining a nucleic acid molecule encoding an extended-PK IL-2 orknottin-Fc mutant and cells transfected with these vectors are among thecertain embodiments.

Vectors suitable for use include T7-based vectors for use in bacteria(see, for example, Rosenberg et al., Gene 56: 125, 1987), the pMSXNDexpression vector for use in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988), and baculovirus-derived vectors (for example theexpression vector pBacPAKS from Clontech, Palo Alto, Calif.) for use ininsect cells. The nucleic acid inserts, which encode the polypeptide ofinterest in such vectors, can be operably linked to a promoter, which isselected based on, for example, the cell type in which expression issought. For example, a T7 promoter can be used in bacteria, a polyhedrinpromoter can be used in insect cells, and a cytomegalovirus ormetallothionein promoter can be used in mammalian cells. Also, in thecase of higher eukaryotes, tissue-specific and cell type-specificpromoters are widely available. These promoters are so named for theirability to direct expression of a nucleic acid molecule in a giventissue or cell type within the body. Skilled artisans are well aware ofnumerous promoters and other regulatory elements which can be used todirect expression of nucleic acids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neon) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that can be used in the invention include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes an extended-PK IL-2 or knottin-Fc mutant are alsofeatures of the invention. A cell of the invention is a transfectedcell, i.e., a cell into which a nucleic acid molecule, for example anucleic acid molecule encoding an extended-PK IL-2 mutant or knottin-Fc,has been introduced by means of recombinant DNA techniques. The progenyof such a cell are also considered within the scope of the invention.

The precise components of the expression system are not critical. Forexample, an extended-PK IL-2 or knottin-Fc mutant can be produced in aprokaryotic host, such as the bacterium E. coli, or in a eukaryotichost, such as an insect cell (e.g., an Sf21 cell), or mammalian cells(e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells areavailable from many sources, including the American Type CultureCollection (Manassas, Va.). In selecting an expression system, itmatters only that the components are compatible with one another.Artisans or ordinary skill are able to make such a determination.Furthermore, if guidance is required in selecting an expression system,skilled artisans may consult Ausubel et al. (Current Protocols inMolecular Biology, John Wiley and Sons, New York, N.Y., 1993) andPouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

Pharmaceutical Compositions and Modes of Administration

In certain embodiments, IL-2 is administered together (simultaneously orsequentially) with a knottin-Fc. In certain embodiments, IL-2 isadministered prior to the administration of a knottin-Fc. In certainembodiments, IL-2 is administered concurrently with the administrationof a knottin-Fc. In certain embodiments, IL-2 is administered subsequentto the administration of a knottin-Fc. In certain embodiments, the IL-2and a knottin-Fc are administered simultaneously. In other embodiments,the IL-2 and a knottin-Fc are administered sequentially. In yet otherembodiments, the IL-2 and a knottin-Fc are administered within one, two,or three days of each other.

In certain embodiments, IL-2 and knottin-Fc are administered with animmune checkpoint blocker. In certain embodiments the immune checkpointblocker is an anti-PD-1 antibody. In certain embodiments, the immunecheckpoint blocker is an anti-CTLA-4 antibody. In certain embodiments,an antagonist of VEGF is used in place of an immune checkpoint blocker.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. Agents include,but are not limited to, in vitro synthetically prepared chemicalcompositions, antibodies, antigen binding regions, and combinations andconjugates thereof. In certain embodiments, an agent can act as anagonist, antagonist, allosteric modulator, or toxin.

In certain embodiments, the invention provides for separatepharmaceutical compositions comprising extended-PK IL-2 with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant, and another pharmaceutical compositioncomprising a knottin-Fc with a pharmaceutically acceptable diluent,carrier, solubilizer, emulsifier, preservative and/or adjuvant. Incertain embodiments, the invention further provides for a separatepharmaceutical composition comprising an immune checkpoint blocker (oran antagonist of VEGF) with a pharmaceutically acceptable diluent,carrier, solubilizer, emulsifier, preservative and/or adjuvant. Incertain embodiments, the pharmaceutical compositions comprise bothextended-PK IL-2 and knottin-Fc with a pharmaceutically acceptablediluents, carrier, solubilizer, emulsifier, preservative and/oradjuvant. In certain embodiments, the pharmaceutical compositioncomprises extended-PK IL-2, knottin-Fc, and an immune checkpoint blocker(or an antagonist of VEGF) with a pharmaceutically acceptable diluents,carrier, solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising extended-PK IL-2, together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant, and another pharmaceutical compositioncomprises a knottin-Fc, together with a pharmaceutically acceptablediluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.In certain embodiments, the invention provides for pharmaceuticalcompositions comprising an immune checkpoint blocker, together with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant. In certain embodiments, each of theagents, e.g., extended-PK IL-2, a knottin-Fc, and optionally an immunecheckpoint blocker (or an antagonist of VEGF), can be formulated asseparate compositions. In certain embodiments, acceptable formulationmaterials preferably are nontoxic to recipients at the dosages andconcentrations employed. In certain embodiments, the formulationmaterial(s) are for s.c. and/or I.V. administration. In certainembodiments, the pharmaceutical composition can contain formulationmaterials for modifying, maintaining or preserving, for example, the pH,osmolality, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. In certain embodiments, suitable formulation materialsinclude, but are not limited to, amino acids (such as glycine,glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants(such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite);buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates orother organic acids); bulking agents (such as mannitol or glycine);chelating agents (such as ethylenediamine tetraacetic acid (EDTA));complexing agents (such as caffeine, polyvinylpyrrolidone,beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers;monosaccharides; disaccharides; and other carbohydrates (such asglucose, mannose or dextrins); proteins (such as serum albumin, gelatinor immunoglobulins); coloring, flavoring and diluting agents;emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone);low molecular weight polypeptides; salt-forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);solvents (such as glycerin, propylene glycol or polyethylene glycol);sugar alcohols (such as mannitol or sorbitol); suspending agents;surfactants or wetting agents (such as pluronics, PEG, sorbitan esters,polysorbates such as polysorbate 20, polysorbate 80, triton,tromethamine, lecithin, cholesterol, tyloxapal); stability enhancingagents (such as sucrose or sorbitol); tonicity enhancing agents (such asalkali metal halides, preferably sodium or potassium chloride, mannitolsorbitol); delivery vehicles; diluents; excipients and/or pharmaceuticaladjuvants. (Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R.Gennaro, ed., Mack Publishing Company (1995). In certain embodiments,the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, the optimalpharmaceutical composition will be determined by one skilled in the artdepending upon, for example, the intended route of administration,delivery format and desired dosage. See, for example, Remington'sPharmaceutical Sciences, supra. In certain embodiments, suchcompositions may influence the physical state, stability, rate of invivo release and rate of in vivo clearance of extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF).

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In certainembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can furtherinclude sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising extended-PK IL-2, a knottin-Fc,and optionally an immune checkpoint blocker (or an antagonist of VEGF),can be prepared for storage by mixing the selected composition havingthe desired degree of purity with optional formulation agents(Remington's Pharmaceutical Sciences, supra) in the form of alyophilized cake or an aqueous solution. Further, in certainembodiments, a composition comprising extended-PK IL-2, a knottin-Fc,and optionally an immune checkpoint blocker (or an antagonist of VEGF),can be formulated as a lyophilizate using appropriate excipients such assucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desiredextended-PK IL-2, a knottin-Fc, and optionally an immune checkpointblocker (or an antagonist of VEGF), in a pharmaceutically acceptablevehicle. In certain embodiments, a vehicle for parenteral injection issterile distilled water in which extended-PK IL-2, a knottin-Fc, andoptionally an immune checkpoint blocker (or an antagonist of VEGF), areformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that can provide for thecontrolled or sustained release of the product which can then bedelivered via a depot injection. In certain embodiments, hyaluronic acidcan also be used, and can have the effect of promoting sustainedduration in the circulation. In certain embodiments, implantable drugdelivery devices can be used to introduce the desired molecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, extended-PK IL-2, a knottin-Fc,and optionally an immune checkpoint blocker (or an antagonist of VEGF),can be formulated as a dry powder for inhalation. In certainembodiments, an inhalation solution comprising extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF), can be formulated with a propellant for aerosoldelivery. In certain embodiments, solutions can be nebulized. Pulmonaryadministration is further described in PCT application No.PCT/US94/001875, which describes pulmonary delivery of chemicallymodified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF), that is administered in this fashion can beformulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Incertain embodiments, a capsule can be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. In certain embodiments, at least one additional agent can beincluded to facilitate absorption of extended-PK IL-2, a knottin-Fc, andoptionally an immune checkpoint blocker (or an antagonist of VEGF). Incertain embodiments, diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of extended-PK IL-2, a knottin-Fc, and optionally animmune checkpoint blocker (or an antagonist of VEGF), in a mixture withnon-toxic excipients which are suitable for the manufacture of tablets.In certain embodiments, by dissolving the tablets in sterile water, oranother appropriate vehicle, solutions can be prepared in unit-doseform. In certain embodiments, suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF), in sustained- or controlled-delivery formulations.In certain embodiments, techniques for formulating a variety of othersustained- or controlled-delivery means, such as liposome carriers,bio-erodible microparticles or porous beads and depot injections, arealso known to those skilled in the art. See for example, PCT ApplicationNo. PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositions canalso include liposomes, which can be prepared by any of several methodsknown in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising extended-PK IL-2 and/or one or morepharmaceutical compositions comprising a knottin-Fc, and optionally animmune checkpoint blocker (or an antagonist of VEGF), to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which extended-PK IL-2, a knottin-Fc, andoptionally an immune checkpoint blocker (or an antagonist of VEGF), arebeing used, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. In certain embodiments, a typical dosage of extended-PK IL-2 anda knottin-Fc can each range from about 0.1 μg/kg to up to about 100mg/kg or more, depending on the factors mentioned above. In certainembodiments, the dosage can range from 0.1 μg/kg up to about 100 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 100 mg/kg.

In certain embodiments, a typical dosage for an immune checkpointblocker can range from about 0.1 mg/kg to up to about 300 mg/kg or more,depending on the factors mentioned above. In certain embodiments, thedosage can range from 1 mg/kg up to about 300 mg/kg; or 5 mg/kg up toabout 300 mg/kg; or 10 mg/kg up to about 300 mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of extended-PK IL-2, a knottin-Fc, andoptionally an immune checkpoint blocker (or an antagonist of VEGF), inthe formulation used. In certain embodiments, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. In certain embodiments, the composition can therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. In certain embodiments, appropriate dosages can beascertained through use of appropriate dose-response data. As describedin the Examples below, dosing can occur daily, every other day, orweekly, all with similar anti-tumor responses.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In certain embodiments, it can be desirable to use apharmaceutical composition comprising extended-PK IL-2, a knottin-Fc,and optionally an immune checkpoint blocker (or an antagonist of VEGF),in an ex vivo manner. In such instances, cells, tissues and/or organsthat have been removed from the patient are exposed to a pharmaceuticalcomposition comprising extended-PK IL-2, a knottin-Fc, and optionally animmune checkpoint blocker (or an antagonist of VEGF), after which thecells, tissues and/or organs are subsequently implanted back into thepatient.

In certain embodiments, extended-PK IL-2, a knottin-Fc, and optionallyan immune checkpoint blocker (or an antagonist of VEGF), can bedelivered by implanting certain cells that have been geneticallyengineered, using methods such as those described herein, to express andsecrete the polypeptides. In certain embodiments, such cells can beanimal or human cells, and can be autologous, heterologous, orxenogeneic. In certain embodiments, the cells can be immortalized. Incertain embodiments, in order to decrease the chance of an immunologicalresponse, the cells can be encapsulated to avoid infiltration ofsurrounding tissues. In certain embodiments, the encapsulation materialsare typically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Methods of Treatment

The extended-PK IL-2, a knottin-Fc, and optionally an immune checkpointblocker (or an antagonist of VEGF), and/or nucleic acids expressingthem, described herein, are useful for treating a disorder associatedwith abnormal apoptosis or a differentiative process (e.g., cellularproliferative disorders or cellular differentiative disorders, such ascancer). Non-limiting examples of cancers that are amenable to treatmentwith the methods of the present invention are described below.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver.Accordingly, the compositions used herein, comprising, e.g., extended-PKIL-2, a knottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF), can be administered to a patient who has cancer.

As used herein, we may use the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” to refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth). Hyperproliferativeand neoplastic disease states may be categorized as pathologic (i.e.,characterizing or constituting a disease state), or they may becategorized as non-pathologic (i.e., as a deviation from normal but notassociated with a disease state). The terms are meant to include alltypes of cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair.

The terms “cancer” or “neoplasm” are used to refer to malignancies ofthe various organ systems, including those affecting the lung, breast,thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, andthe genitourinary tract, as well as to adenocarcinomas which aregenerally considered to include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The mutant IL-2 polypeptidescan be used to treat patients who have, who are suspected of having, orwho may be at high risk for developing any type of cancer, includingrenal carcinoma or melanoma, or any viral disease. Exemplary carcinomasinclude those forming from tissue of the cervix, lung, prostate, breast,head and neck, colon and ovary. The term also includes carcinosarcomas,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias (e.g., erythroblasticleukemia and acute megakaryoblastic leukemia). Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macro globulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

It will be appreciated by those skilled in the art that amounts for eachof the extended-PK IL-2, a knottin-Fc, and optionally an immunecheckpoint blocker (or an antagonist of VEGF), that are sufficient toreduce tumor growth and size, or a therapeutically effective amount,will vary not only on the particular compounds or compositions selected,but also with the route of administration, the nature of the conditionbeing treated, and the age and condition of the patient, and willultimately be at the discretion of the patient's physician orpharmacist. The length of time during which the compounds used in theinstant method will be given varies on an individual basis.

It will be appreciated by those skilled in the art that the B16 melanomamodel used herein is a generalized model for solid tumors. That is,efficacy of treatments in this model is also predictive of efficacy ofthe treatments in other non-melanoma solid tumors. For example, asdescribed in Baird et al. (J Immunology 2013; 190:469-78; Epub Dec. 7,2012), efficacy of cps, a parasite strain that induces an adaptiveimmune response, in mediating anti-tumor immunity against B16F10 tumorswas found to be generalizable to other solid tumors, including models oflung carcinoma and ovarian cancer. In another example, results from aline of research into VEGF targeting lymphocytes also shows that resultsin B16F10 tumors were generalizable to the other tumor types studied(Chinnasamy et al., JCI 2010; 120:3953-68; Chinnasamy et al., ClinCancer Res 2012; 18:1672-83). In yet another example, immunotherapyinvolving LAG-3 and PD-1 led to reduced tumor burden, with generalizableresults in a fibrosarcoma and colon adenocarcinoma cell lines (Woo etal., Cancer Res 2012; 72:917-27).

In certain embodiments, the extended-PK IL-2, knottin-Fc, and optionalimmune checkpoint blocker (or an antagonist of VEGF), are used to treatcancer.

In certain embodiments, the extended-PK IL-2, knottin-Fc, and optionalimmune checkpoint blocker (or an antagonist of VEGF) are used to treatmelanoma, leukemia, lung cancer, breast cancer, prostate cancer, ovariancancer, colon cancer, renal carcinoma, and brain cancer.

In certain embodiments, the extended-PK IL-2, knottin-Fc and optionalimmune checkpoint blocker (or an antagonist of VEGF) inhibit growthand/or proliferation of tumor cells.

In certain embodiments, the extended-PK IL-2, knottin-Fc, and optionalimmune checkpoint blocker (or an antagonist of VEGF) reduce tumor size.

In certain embodiments, the extended-PK IL-2, knottin-Fc, and optionalimmune checkpoint blocker (or an antagonist of VEGF) inhibit metastasesof a primary tumor.

In certain embodiments, knottin-Fc and an immune checkpoint blocker (oran antagonist of VEGF), with or without IL-2, inhibit growth and/orproliferation of tumor cells. In certain embodiments, knottin-Fc and animmune checkpoint blocker (or an antagonist of VEGF), with or withoutIL-2, reduce tumor size. In certain embodiments, knottin-Fc and animmune checkpoint blocker, with or without IL-2, inhibit metastases of aprimary tumor.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment of thenoted cancers and symptoms.

Kits

A kit can include extended-PK IL-2, a knottin-Fc, and optionally animmune checkpoint blocker (or an antagonist of VEGF), as disclosedherein, and instructions for use. The kits may comprise, in a suitablecontainer, extended-PK IL-2, an integrin binding knottin-Fc, an optionalimmune checkpoint blocker (or an antagonist of VEGF), one or morecontrols, and various buffers, reagents, enzymes and other standardingredients well known in the art. Certain embodiments include a kitwith extended-PK IL-2, knottin-Fc, and optional immune checkpointblocker (or an antagonist of VEGF) in the same vial. In certainembodiments, a kit includes extended-PK IL-2, knottin-Fc, and optionalimmune checkpoint blocker (or an antagonist of VEGF) in separate vials.

The container can include at least one vial, well, test tube, flask,bottle, syringe, or other container means, into which extended-PK IL-2,a knottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF) may be placed, and in some instances, suitablyaliquoted. Where an additional component is provided, the kit cancontain additional containers into which this component may be placed.The kits can also include a means for containing extended-PK IL-2, aknottin-Fc, and optionally an immune checkpoint blocker (or anantagonist of VEGF) and any other reagent containers in closeconfinement for commercial sale. Such containers may include injectionor blow-molded plastic containers into which the desired vials areretained. Containers and/or kits can include labeling with instructionsfor use and/or warnings.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures of PCTpublication WO 13/177187, U.S. Pat. No. 8,536,301, and U.S. PatentPublication No. 2014/0073518 are expressly incorporated herein byreference.

EXAMPLES

Below are examples of specific embodiments for carrying out the methodsdescribed herein. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperatures, etc.), but some experimentalerror and deviation should, of course, be allowed for. The practice ofthe present invention will employ, unless otherwise indicated,conventional methods of protein chemistry, biochemistry, recombinant DNAtechniques and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., T. E.Creighton, Proteins: Structures and Molecular Properties (W.H. Freemanand Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers,Inc., current addition); Sambrook, et al., Molecular Cloning: ALaboratory Manual (2^(nd) Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18^(th) Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Moreover, while the examples below employ extended-PK IL-2 of mouseorigin (i.e., both the extended-PK group (mouse serum albumin) and IL-2are of mouse origin) and mouse Fc fused to an integrin-binding knottin,it should be understood that corresponding human extended-PK IL-2 (i.e.,human serum albumin (HSA) and human IL-2, and variants thereof) andintegrin-binding-human-Fc (i.e., Fc from human IgG1 fused to knottin)can be readily generated by those of ordinary skill in the art usingmethods described supra, and used in the methods disclosed herein.

Example 1 Knottin-Fc Treatment is as Effective as TA99 Treatment inB16F10 Tumors

To address the lack of general tumor-associated antigens, a knottin-Fcprotein was engineered. The knottin-Fc protein comprises two parts: 1)an engineered cystine knot (knottin) peptide that binds with highaffinity to tumor-associated αvβ3, αvβ5, and α5β1 integrin receptors(specifically 2.5F, SEQ ID NO: 94 or 96), and 2) an antibody Fc domainthat mediates immune effector functions in vivo. The knottin-Fc used is2.5F with a KISS substitution, fused to a mouse IgG2a Fc domain, SEQ IDNO: 45, unless stated otherwise.

To determine the effects of the knottin-Fc on tumor growth, 2.5×10⁵B16F10 murine melanoma cells were injected into the flanks of C57BL/6mice subcutaneously. Prophylactic treatment was done with 80 μgknottin-Fc, 80 μg knottin-D265A (The D265A mutation in the murine IgG2aFc domain eliminates binding to FcγR and complement), or 200 μg TA99,administered intraperitoneally every two days, starting on the day oftumor inoculation, for a total of ten treatments. TA99 is an antibodywhich binds to TYRP-1, (an antigen found on melanoma cells) and inhibitsmelanoma tumor growth.

Tumor area was measured and plotted (FIG. 2). Tumor area was measuredusing calipers. The longest dimension of the tumor in any direction wasmeasured first, followed by the measurement of the longest perpendiculardimension. The two values were then multiplied to quantify tumor area insquare millimeters.

Both the TA99 treatment and the knottin-Fc treatment controlled tumorgrowth to a similar extent. The failure of knottin-D265A to preventtumor growth indicates that tumor control by knottin-Fc is mediated bythe effector function of Fc.

Example 2 Knottin-Fc and Extended-PK IL-2 Synergistically Control TumorGrowth of B16F10 Tumors

A therapeutic study was conducted to examine the effect of addingMSA/IL-2 to knottin-Fc treatment. Tumors were established by injectingC57BL/6 mice subcutaneously with 1×10⁶ B16F10 murine melanoma cells. 30μg MSA/IL-2 and/or 500 μg knottin-Fc was administered intraperitoneallyon day 6 after tumor inoculation and every 6 days after that for 4treatments total.

Tumor area was measured and plotted (FIG. 3A). While MSA/IL-2 andknottin-Fc alone had no effect on tumor growth, the combination ofMSA/IL-2 and knottin-Fc effectively controlled tumor growth. AKaplan-Meier survival plot revealed that the combination of MSA/IL-2 andknottin-Fc extended the survival of these mice, whereas monotherapieshad no significant effect (FIG. 3B).

Example 3 Knottin-Fc and Extended-PK IL-2 Synergistically Control TumorGrowth of MC38 Tumors

A separate therapeutic study was conducted to examine the effect ofMSA/IL-2 on knottin-Fc efficacy in a tumor type that has no reportedtargetable antigens. 1×10⁶ MC38 murine colon carcinoma cells wereinjected into the flanks of C57BL/6 mice. 6 days after tumor inoculationand every 6 days after for a total of 4 treatments, 30 μg MSA-IL-2and/or 500 μg knottin-Fc was administered intraperitoneally.

Tumor growth was controlled with the combination of MSA/IL-2 andknottin-Fc, but not when each component was administered as amonotherapy (FIG. 4A). A Kaplan-Meier survival plot shows an increase insurvival in the mice treated with the combination of MSA/IL-2 andknottin-Fc compared to when each component was administered as amonotherapy (FIG. 4B).

Example 4 Knottin-Fc and Extended-PK IL-2 Synergistically Control TumorGrowth of Ag104A Tumors

An additional study was conducted to examine the effects of MSA/IL-2 onknottin-Fc efficacy in a different tumor type. Ag104A fibrosarcomatumors were established by injecting 1.0×10⁶ Ag104A cells into theflanks of C3H/HeN mice. Starting six days after tumor inoculation andevery 6 days after, 30 μg MSA/IL-2 and/or 500 μg knottin-Fc wasadministered intraperitoneally.

Tumor growth was controlled with the combination of MSA/IL-2 andknottin-Fc, but not when each component was administered as amonotherapy (FIG. 5A). A Kaplan-Meier survival plot shows an increase insurvival in mice treated with the combination of MSA/IL-2 and knottin-Fccompared to when each component was administered alone (FIG. 5B).

Example 5 Effector Function is Required for Synergistic Tumor Control

The results of Example 1 indicate that effector function is required fortumor control by knottin-Fc. To determine the role of effector functionin tumor control by synergistic treatment with MSA/IL-2, a further studywas conducted using both B16F10 and MC38 cells. 1×10⁶ B16F10 cells(FIGS. 6A and 6B) or MC38 cells (FIGS. 7A and 7B) were injected into theflanks of C57BL/6 mice. 30 μg MSA-IL-2 was administeredintraperitoneally on day 6 after tumor inoculation and every 6 daysafter that for 4 treatments total. 500 μg knottin-Fc or knottin-D265AFc(the D265A mutation in the murine IgG2a Fc domain eliminates binding toFcγR and complement) was administered intraperitoneally on day 6 aftertumor inoculation and every six days thereafter for a total of 4treatments.

Individual tumor size measurements (FIGS. 6A and 7A) and survival plots(FIGS. 6B and 7B) indicate that tumor control by knottin-Fc requires theeffector function of Fc.

Example 6 Therapeutic Antibody and Immune Checkpoint Blocker Enhance theEfficacy of Knottin-Fc and Extended-PK IL-2 in B16F10 Tumors

To determine the effect of antibodies on knottin-Fc and extended-PK IL-2synergistic tumor control, an additional study was carried out usingB16F10 cells. 1×10⁶ B16F10 cells were injected into the flanks ofC57BL/6 mice. 30 μg MSA/IL-2 was administered every 6 days beginning onday 6 after tumor inoculation for a total of 5 treatments and 200 μgknottin-Fc was administered daily from days 6-30 after tumorinoculation. Antibodies against TYRP-1 (TA99) were administered at 100μg per mouse every 6 days starting on day 6 after B16F10 tumorinoculation. Antibodies against PD-1 were administered at 200 μg permouse every 6 days starting on day 6 after tumor inoculation.

Individual tumor size measurements (FIG. 8A) and a survival plot (FIG.8B) indicate that antibodies enhanced tumor control via knottin-Fc andMSA/IL-2. However, this is only effective if the antibody targets anantigen present on the tumor cells. Combining knottin-Fc and MSA/IL-2with a therapeutic antibody or immune checkpoint blocker increased theefficacy of tumor control and survival improvement.

Example 7 Immune Checkpoint Blocker Enhances the Efficacy of Knottin-Fcand Extended-PK IL-2 in MC38 Tumors

To determine the effect of antibodies on knottin-Fc and extended-PK IL-2synergistic tumor control, an additional study was carried out usingMC38 cells. 1×10⁶ MC38 cells were injected into the flanks of C57BL/6mice. 30 μg MSA/IL-2 and 500 μg knottin-Fc were administered every 6days beginning on day 6 after tumor inoculation for a total of 4treatments. Antibodies against PD-1 were administered at 200 μg permouse every 6 days starting on day 6 after tumor inoculation.

Individual tumor size measurements (FIG. 9A) and survival plots (FIG.9B) indicate that antibodies enhanced tumor control via knottin-Fc andMSA/IL-2. However, this is only effective if the antibody targets anantigen present on the tumor cells. The most effective treatment was tocombine knottin-Fc and MSA/IL-2 with an immune checkpoint blockade(anti-PD-1) antibody.

Example 8 Knottin-Fc is Effective when Administered Every Other Day orWeekly

To determine the minimum dosing requirement for an anti-tumor response,the B16F10 melanoma and MC38 colon carcinoma tumor models were used andknottin-Fc was administered daily, every other day, or weekly. 1×10⁶B16F10 cells (FIG. 10) or 1×10⁶ MC38 cells (FIG. 11) were injected intothe flanks of C57BL/6 mice. 30 μg of MSA/IL-2 was administered on days6, 12, 18, and 24 after tumor inoculation. 200 μg of knottin-Fc wasadministered daily or every other day starting on day 6 and ending onday 28. 500 μg of knottin-Fc was administered on days 6, 12, 18, and 24for the weekly regimen. In both models, administration of knottin-Fc inconjunction with MSA/IL-2 every other day or every 6 days was equivalentto daily administration.

Example 9 Knottin-Fc and Fc-Knottin Constructs Control Tumor Growth

The determine the optimal placement of Fc fused to knottin for ananti-tumor response, Fc was fused to either the N-terminus (Knottin-Fc)or the C-terminus (Fc-Knottin). 2.5×10⁵ B16F10 melanoma cells wereinjected into the flanks of C57BL/6 mice. 80 μg of knottin-Fc, 80 μg ofFc-knottin, or 200 μg of IgG-knottin was administered immediately aftertumor inoculation and every two days after, for a total of 10treatments. Tumor area was measured (FIG. 12). There was essentially nodifference in efficacy between the different knottin-Fc fusion proteins.

To validate this finding, another mouse tumor model was utilized. 1×10⁶MC38 colon carcinoma cells were injected into the flanks of albinoC57BL/6 mice. 200 μg of knottin-Fc or Fc-knottin were administeredimmediately after tumor inoculation and every two days after, for atotal of 10 treatments. Tumor area was measured (FIG. 13). There wasessentially no difference in efficacy between knottin-Fc and Fc-knottin.The results from both of these mouse tumor models indicate that theplacement of Fc in the knottin-Fc fusion protein does not affectefficacy for an anti-tumor response.

To determine optimal integrin-binding-knottin-Fc constructs, a fusionprotein was made containing a (Gly4Ser)3 linker in between the knottinand the Fc domain. No difference in integrin binding affinity to U87MGglioblastoma cells were observed compared to integrin-binding-knottin-Fccontaining no linker (data not shown).

Example 10 Knottin-Fc and IL-2 Combination Protects Against SecondaryTumor Challenge

To determine if the combination of knottin-Fc and MSA/IL-2 could protecttreated mice from a secondary challenge, the MC38 tumor model was used.1×10⁶ MC38 cells were injected into the flanks of C57BL/6 mice and both30 μg MSA/IL-2 and 500 μg knottin-Fc were administered every 6 daysbeginning on day 6 after tumor inoculation for a total of 4 treatments.16-20 weeks after the initial tumor inoculation, previously cured miceor age-matched naïve mice were inoculated with 1×10⁶ MC38 cells in theopposite flank. No further treatment was administered.

Individual tumor size measurements (FIG. 14A) and survival plot (FIG.14B) indicate that knottin-Fc and MSA/IL-2 protect previously treatedmice against secondary tumor challenge, demonstrating a potent andsustained immune response against the tumors.

Example 11 Knottin-Fc Targeting All Three Integrins Most EffectivelyControls Tumor Growth

To assess the efficacy of a knottin-Fc that targets all three integrins(i.e., αvβ3, αvβ5, and α5β1, “2.5F_knottin-Fc” SEQ ID NO: 45) comparedto a knottin-Fc that targets only two integrins (i.e., αvβ3 and αvβ5,“2.5D_knottin-Fc” SEQ ID NO: 47) in controlling tumor growth, anexperiment was conducted using three different tumors models (i.e.,B16F10, MC38, and Ag104A). 1×10⁶ B16F10 (FIGS. 15A and 15B) or MC38cells (FIGS. 16A and 16B) were injected into the flanks of C57BL/6 mice.1.0×10⁶ Ag104A cells (FIGS. 17A and 17B) were injected into the flanksof C3H/HeN mice. 30 μg MSA/IL-2, 500 μg 2.5F_knottin-Fc and/or2.5D_knottin-Fc were administered every 6 days beginning on day 6 aftertumor inoculation for a total of 4 treatments.

Individual tumor size measurements (FIGS. 15A, 16A and 17A) and survivalplots (FIGS. 15B, 16B and 17B) indicate that a knottin-Fc that targetsall three integrins more effectively controls tumor growth than aknottin-Fc that only targets two integrins.

Example 12 Immune Cell Populations Important for Survival inTumor-Bearing Mice

To assess the role of various immune cell populations in the improvedsurvival of mice with MC38 tumors treated with knottin-Fc and MSA/IL-2,depletion experiments were conducted. 400 μg anti-CD8, anti-CD4,anti-NK1.1, anti-Ly6G or anti-CD19 antibodies were administered everyfour days for a total of six treatments starting on day 4 after tumorinoculation. 300 μg anti-CSF-1R antibody was administered every two daysfor a total of eleven treatments starting on day 4 of tumor inoculation.30 μg cobra venom factor (CVF) was administered every 6 days for a totalof 4 treatments starting on day 5 after tumor inoculation. Anti-CD8depletes CD8+ T cells, anti-CD4 depletes CD4+ T cells, anti-NK1.1depletes natural killer cells, anti-Ly6G depletes neutrophils, anti-CD19depletes B cells, and anti-CSF-1R depletestissue-resident/tumor-resident macrophages. CVF inhibits complementactivity.

Individual tumor size measurements (FIG. 18A) and survival plots (FIG.18B) indicate that CD8+ T cells and macrophages are particularlyimportant for the synergistic effect of knottin-Fc and MSA/IL-2 on tumorcontrol.

In addition to the immune cell depletions, Batf3−/− mice were used toevaluate the role of cross-presenting CD8+ dendritic cells. Batf3−/−mice lack the function of the basic leucine zipper transcription factor,ATF-like 3. Deletion of Batf3 has been shown to prevent the developmentof CD8+ dendritic cells, which are important for the cross-presentationof exogenous antigen on MHC Class I.

Tumor areas of treated C57BL/6 (i.e. “control”) and Batf3−/− mice weremeasured and plotted (FIG. 19A), and demonstrated less tumor control inBatf3−/− mice treated with MSA/IL-2 and knottin-Fc. A Kaplan-Meiersurvival plot was also generated, and revealed a significant decrease insurvival in Batf3−/− mice treated with knottin-Fc and MSA/IL-2 comparedto C57BL/6 mice (FIG. 19B). This indicated that cross-presentation bydendritic cells may be a factor that contributes to the efficacy of thecombination therapy on controlling the growth of MC38 tumors.

Since CD8+ T cells were found to be critical for efficacy of theknottin-Fc and MSA-IL-2 combination, and IFNγ was previously reported tobe important in the efficacy of MSA-IL-2 and therapeutic antibodycombinations (Zhu et al., Cancer Cell (2015) Vol 27: 489-501), the roleof IFNγ was investigated in the MC38 tumor model. 1×10⁶ MC38 wereinjected into the flanks of C57BL/6 mice. 30 μg MSA/IL-2 and 500 μgknottin-Fc were administered every 6 days beginning on day 6 after tumorinoculation for a total of 4 treatments. 200 μg of anti-INFγ antibodywas administered every two days for a total of eleven treatmentsstarting on day 5 after rumor inoculation.

As shown in FIGS. 20A and 20B, IFNγ depletion did not significantlyaffect tumor control or survival of mice with MC38 tumors.

Example 13 Antagonizing Immune Suppression Improves Survival ofTumor-Bearing Mice

To determine whether the efficacy of tumor control could be enhanced byinhibiting suppressors of the immune system, an antagonist of vascularendothelial growth factor (VEGF) or cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4) was administered in mice with MC38 or B16F10 tumors.1×10⁶ MC38 cells (FIGS. 21A, 21B and 22) or B16F10 (FIGS. 23A and 23B)were injected into the flanks of C57BL/6 mice. 30 μg MSA/IL-2, 500 μgknottin-Fc, and 200 μg anti-VEGF antibody or anti-CTLA-4 antibody wereadministered every 6 days beginning on day 6 after tumor inoculation fora total of 4 treatments.

Synthetic genes, encoding the heavy (VH) and light (VL) chain variableregions of anti-VEGF (clone B20-4.1.1) (Bagri et al., Clin. Cancer Res.(2010) Vol 16: 3887-900) and anti-CTLA-4 (9D9) (Selby et al., CancerImmunol. Res. (2013) Vol 1:32-42) antibodies, were codon-optimized forexpression in mammalian cells (GeneArt, Life Technologies). B20-4.1.1antibody constructs were generated by PCR-amplification of DNA insertsencoding for VH and VL regions and cloned via Gibson assembly intoseparate gWiz expression vectors (Genlantis) containing a CMV promoter,a kanamycin antibiotic resistance gene and the DNA sequence encoding foreither murine IgG2a heavy-chain (CH1, CH2 and CH3) or light-chain (CL)constant regions (see below Sequence 1 and 2 for B20-4.1.1). The 9D9antibody construct was generated by PCR-amplification of DNA insertsencoding the VH and VL regions and sub-cloned into a double cassettep2MPT expression vector (EPFL Protein expression core facility,http://pecf.epfl.ch), containing a CMV promoter, an ampicillinantibiotic resistance gene and both murine IgG2a heavy-chain (CH1, CH2and CH3) or light-chain (CL) constant regions, via the restriction sitesNotI/BamHI and EcoRI/XbaI, respectively. All constructs were verified byDNA sequencing (Macrogen).

Anti-VEGF antibody B20-4.1.1 was expressed in transiently transfectedhuman embryonic kidney (HEK293-F) cells using the Free-Style 293Expression System (Life Technologies) as described previously (Zhu etal., Cancer Cell (2015) Vol 27: 489-501). Antibody 9D9 was expressed intransiently transfected Chinese hamster ovary (CHO) cells and providedby EPFL Protein expression core facility (http://pecf.epfl.ch). After 7days of expression, cells were removed by centrifugation (15,000×g for30 min at 4° C.) and filtration (0.22 μm PES membranes filter) and theproteins in the supernatant purified by protein A chromatographyaccording to the manufacturer's instructions (GE Healthcare). Elutedantibodies were further desalted and purified on a HiLoad Superdex 20010/600 size exclusion column (GE Healthcare) connected to anAKTApurifier system and equilibrated with buffer PBS 1× pH 7.4. Thepurified antibodies were concentrated using a 30000 NMWL Amicon Ultracentrifugal filter device (Millipore) at 4000 g and 4° C. and quantifiedby measuring absorbance at 280 nm using a NanoDrop 2000spectrophotometer (Thermo Scientific). Molecular weights were confirmedby reducing and non-reducing SDS/PAGE. Purity was evaluated by FPLC.Protein samples were analyzed by SDS-PAGE under denaturating andreducing conditions using NuPAGE 4-12% Bis-Tris Gels (Life Technologies)in MOPS buffer followed by Coomassie staining. Native size andoligomerization state of antibodies after concentration were alsoanalyzed by size-exclusion chromatography with a Superdex 200 10/300 GLcolumn (GE Healthcare) connected to an AKTApurifier system andequilibrated with buffer PBS 1× pH 7.4.

Tumor area was measured and plotted (FIGS. 21A, 22 and 23A) and survivalplots were generated (FIGS. 21B and 23B). The triple combinations (i.e.,MSA/IL-2+knottin-Fc+anti-VEGF and MSA/IL-2+knottin-Fc+anti-CTLA-4)significantly controlled tumor growth and improved survival compared tothe double combinations (i.e., MSA/IL-2+anti-VEGF andknottin-Fc+anti-VEGF or MSA/IL-2+anti-CTLA-4 andknottin-Fc+anti-CTLA-4). This is similar to the results observed whenadding an anti-PD-1 antibody to the combination of MSA/IL-2 andknottin-Fc, which indicated that other suppressors of the immune systemcould be inhibited to improve the efficacy of the combination ofMSA/IL-2+knottin-Fc. The improved therapeutic efficacy by addition of ananti-VEGF antibody was also observed in RENCA (renal adenocarcinoma) andLLC (Lewis lung carcinoma) tumor models (data not shown).

EQUIVALENTS

Those skilled in the art will recognize or be able to ascertain, usingno more than routine experimentation, many equivalents of the specificembodiments described herein described herein. Such equivalents areintended to be encompassed by the following claims.

TABLE 2 Sequence Summary SEQ ID NO Description Sequence 1 HumanASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL IgG1TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT constantKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR regionTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST (amino acidYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR sequence)EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 2 HumanEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV IgG1 FcVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV domainLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT (amino acidLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP sequence)VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK 3 HumanDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV IgG1 FcSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH domainQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR (amino acidDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS sequence)DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP Deletion GK (ΔEPKSC) UpperHinge 4 Mouse IL-2 GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC (nucleicAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG acidAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGA sequence)GAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 5Mouse IL-2 APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN (amino acidYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQS sequence)KSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRR WIAFCQSIISTSPQ 6 QQ6210GCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC (nucleicAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG acidAGCAGCTGTTGATGGACCTACAGGAACTCCTGAGTAGGATGGA sequence)GGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCAT CTCAACAAGCCCTCAA 7 QQ6210APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMED (amino acidHRNLRLPRMLTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSK sequence)SFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRW IAFCQSIISTSPQ 8 E76AGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC (nucleicAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG acidAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGA sequence)GAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 9 E76AAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN (amino acidYRNLKLPRMLTFKFYLPKQATELKDLQCLEDALGPLRHVLDLTQS sequence)KSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRR WIAFCQSIISTSPQ 10 E76GGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC (nucleicAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG acidAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGA sequence)GAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATC TCAACAAGCCCTCAA 11 E76GAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEN (amino acidYRNLKLPRMLTFKFYLPKQATELKDLQCLEDGLGPLRHVLDLTQS sequence)KSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRR WIAFCQSIISTSPQ 12 D265AATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT Fc/FlagCCCAGGTGCACGATGTGAGCCCAGAGTGCCCATAACACAGAAC (nucleicCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGA acidCCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCA sequence)AGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTG (C-terminalGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCT flag tag isGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAAC underlined)CCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGTGGCGGATCTGACTACAAGGACGACGATGACAAGTGATAA 13 D265AMRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLL Fc/FlagGGPSVFIFPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNN (amino acidVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN sequence)RALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF (C-terminalLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST flag tag isWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSDYKDDDDK underlined) 14 D265AATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT Fc/wt mIL-2CCCAGGTGCACGATGTGAGCCCAGAGTGCCCATAACACAGAAC (nucleicCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGA acidCCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCA sequence)AGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTG (C-terminalGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCT 6x his tag isGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAAC underlined)CCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATA A 15 D265AMRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLL Fc/wt mIL-2GGPSVFIFPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNN (amino acidVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN sequence)RALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF (C-terminalLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST 6x his tag isWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSST underlined)AEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTS PQHHHHHH** 16 D265A Fc/ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT QQ6210CCCAGGTGCACGATGTGAGCCCAGAGTGCCCATAACACAGAAC (nucleicCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGA acidCCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCA sequence)AGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTG (C-terminalGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCT 6x his tag isGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAAC underlined)CCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAACTCCTGAGTAGGATGGAGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATA A 17 D265A Fc/MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLL QQ6210GGPSVFIFPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNN (amino acidVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN sequence)RALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF (C-terminalLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST 6x his tag isWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSST underlined)AEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDHRNLRLPRMLTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQSIISTSP QHHHHHH 18 D265A Fc /ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT E76ACCCAGGTGCACGATGTGAGCCCAGAGTGCCCATAACACAGAAC (nucleicCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGA acidCCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCA sequence)AGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTG (C-terminalGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCT 6x his tag isGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAAC underlined)CCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGCTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATA A 19 D265A Fc /MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLL E76AGGPSVFIFPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNN (amino acidVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN sequence)RALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF (C-terminalLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST 6x his tag isWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSST underlined)AEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDALGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIIST SPQHHHHHH 20 D265A Fc /ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCT E76GCCCAGGTGCACGATGTGAGCCCAGAGTGCCCATAACACAGAAC (nucleicCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGA acidCCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCA sequence)AGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTG (C-terminalGTGGTGGCCGTGAGCGAGGATGACCCAGACGTCCAGATCAGCT 6x his tag isGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAAC underlined)CCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAAGGAGGGGGCTCCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACAGAATTGAAAGATCTTCAGTGCCTAGAAGATGGTCTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAACACCATCACCACCATCACTGATA A 21 D265A Fc /MRVPAQLLGLLLLWLPGARCEPRVPITQNPCPPLKECPPCAAPDLL E76GGGPSVFIFPPKIKDVLMISLSPMVTCVVVAVSEDDPDVQISWFVNN (amino acidVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNN sequence)RALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGF (C-terminalLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKST 6x his tag isWERGSLFACSVVHEGLHNHLTTKTISRSLGKGGGSAPTSSSTSSST underlined)AEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDGLGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIIST SPQHHHHHH 22 mIL-2 QQGCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC 6.2-4AACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG (nucleicAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGA acidGGATTCCAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAA sequence)TTTTACTTGCCCAAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGGCTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCAT CTCAACGAGCCCTCAA 23mIL-2 QQ APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMEDS 6.2-4RNLRLPRMLTFKFYLPKQATELEDLQCLEDELEPLRQVLDLTQSKS (amino acidFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVGFLRRWI sequence) AFCQSIISTSPQ 24mIL-2 QQ GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC 6.2-8AACAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTTGAT (nucleicGGACCTACAGGAGCTCCTGAGTAGGATGGAGGATCACAGGAAC acidCTGAGACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAA sequence)GCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCC TCGA 25 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQHLEQLLMDLQELLSRMEDHRNLR 6.2-8LPRMLTFKFYLPKQATELEDLQCLEDELEPLRQVLDLTQSKSFQLE (amino acidDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRWIAFCQ sequence) SIISTSPR 26mIL-2 QQ GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC 6.2-10AACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG (nucleicAGCAGCTGTTGATGGACCTACAGGAACTCCTGAGTAGGATGGA acidGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAA sequence)TTTTACTTGCCCGAGCAGGCCACAGAATTGGAAGATCTTCAGTGCCTAGAAGATGAACTTGAACCACTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCAT CTCAACAAGCCCTCAG 27mIL-2 QQ APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMED 6.2-10HRNLRLPRMLTFKFYLPEQATELEDLQCLEDELEPLRQVLDLTQSK (amino acidSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRW sequence) IAFCQSIISTSPQ28 mIL-2 QQ GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC 6.2-11AACAGCAGCAGCAGCAGCAGCAGCAGCACCTGGAGCAGCTGTT (nucleicGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGGATTCCAGG acidAACCTGAGACTCCCCAGAATGCTCACCTTCAAATTTTACTTGCC sequence)CGAGCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAAGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGACGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAG CCCTCAG 29 mIL-2 QQAPTSSSTSSSTAEAQQQQQQQQQHLEQLLMDLQELLSRMEDSRNL 6.2-11RLPRMLTFKFYLPEQATELKDLQCLEDELEPLRQVLDLTQSKSFQL (amino acidEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRRWIAFC sequence) QSIISTSPQ 30mIL-2 QQ GCACCCACCTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCAC 6.2-13AACAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCTGG (nucleicAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGTAGGATGGA acidGGATCACAGGAACCTGAGACTCCCCAGGATGCTCACCTTCAAA sequence)TTTTACTTGCCCGAGCAGGCCACAGAATTGAAAGATCTCCAGTGCCTAGAAGATGAACTTGAACCTCTGCGGCAGGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTGTTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGCCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATC ATCTCAACAAGCCCTCAG 31mIL-2 QQ APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMED 6.2-13HRNLRLPRMLTFKFYLPEQATELKDLQCLEDELEPLRQVLDLTQS (amino acidKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDEPATVVDFLRR sequence) WIAFCQSIISTSPQ32 Full length ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGC human IL-2ACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAA (nucleicACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGA acidTTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAG sequence)GATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA 33 Full lengthMYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILN human IL-2GINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLN (amino acidLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL sequence)NRWITFCQSIISTLT 34 Human IL-2GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGG withoutAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAAT signalAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGT peptideTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTG (nucleicTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTA acidGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCA sequence)GCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCA ACACTGACTTGA 35 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY withoutMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINV signalIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT peptide (amino acid sequence)36 Human MDMRVPAQLLGLLLLWLPGARCADAHKSEVAHRFKDLGEENFK serumALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKS albuminLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN (amino acidPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPEL sequence)LFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGS 37 MatureDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE HSA (aminoVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD acidCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETF sequence)LKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST LTGGGS 38 HumanATGGATATGCGGGTGCCTGCTCAGCTGCTGGGACTGCTGCTGCT serumGTGGCTGCCTGGGGCTAGATGCGCCGATGCTCACAAAAGCGAA albuminGTCGCACACAGGTTCAAAGATCTGGGGGAGGAAAACTTTAAGG (nucleicCTCTGGTGCTGATTGCATTCGCCCAGTACCTGCAGCAGTGCCCC acidTTTGAGGACCACGTGAAACTGGTCAACGAAGTGACTGAGTTCG sequence)CCAAGACCTGCGTGGCCGACGAATCTGCTGAGAATTGTGATAAAAGTCTGCATACTCTGTTTGGGGATAAGCTGTGTACAGTGGCCACTCTGCGAGAAACCTATGGAGAGATGGCAGACTGCTGTGCCAAACAGGAACCCGAGCGGAACGAATGCTTCCTGCAGCATAAGGACGATAACCCCAATCTGCCTCGCCTGGTGCGACCTGAGGTGGACGTCATGTGTACAGCCTTCCACGATAATGAGGAAACTTTTCTGAAGAAATACCTGTACGAAATCGCTCGGAGACATCCTTACTTTTATGCACCAGAGCTGCTGTTCTTTGCCAAACGCTACAAGGCCGCTTTCACCGAGTGCTGTCAGGCAGCCGATAAAGCTGCATGCCTGCTGCCTAAGCTGGACGAACTGAGGGATGAGGGCAAGGCCAGCTCCGCTAAACAGCGCCTGAAGTGTGCTAGCCTGCAGAAATTCGGGGAGCGAGCCTTCAAGGCTTGGGCAGTGGCACGGCTGAGTCAGAGATTCCCAAAGGCAGAATTTGCCGAGGTCTCAAAACTGGTGACCGACCTGACAAAGGTGCACACCGAATGCTGTCATGGCGACCTGCTGGAGTGCGCCGACGATCGAGCTGATCTGGCAAAGTATATTTGTGAGAACCAGGACTCCATCTCTAGTAAGCTGAAAGAATGCTGTGAGAAACCACTGCTGGAAAAGTCTCACTGCATTGCCGAAGTGGAGAACGACGAGATGCCAGCTGATCTGCCCTCACTGGCCGCTGACTTCGTCGAAAGCAAAGATGTGTGTAAGAATTACGCTGAGGCAAAGGATGTGTTCCTGGGAATGTTTCTGTACGAGTATGCCAGGCGCCACCCAGACTACTCCGTGGTCCTGCTGCTGAGGCTGGCTAAAACATATGAAACCACACTGGAGAAGTGCTGTGCAGCCGCTGATCCCCATGAATGCTATGCCAAAGTCTTCGACGAGTTTAAGCCCCTGGTGGAGGAACCTCAGAACCTGATCAAACAGAATTGTGAACTGTTTGAGCAGCTGGGCGAGTACAAGTTCCAGAACGCCCTGCTGGTGCGCTATACCAAGAAAGTCCCACAGGTGTCCACACCCACTCTGGTGGAGGTGAGCCGGAATCTGGGCAAAGTGGGGAGTAAATGCTGTAAGCACCCTGAAGCCAAGAGGATGCCATGCGCTGAGGATTACCTGAGTGTGGTCCTGAATCAGCTGTGTGTCCTGCATGAAAAAACACCTGTCAGCGACCGGGTGACAAAGTGCTGTACTGAGTCACTGGTGAACCGACGGCCCTGCTTTAGCGCCCTGGAAGTCGATGAGACTTATGTGCCTAAAGAGTTCAACGCTGAGACCTTCACATTTCACGCAGACATTTGTACCCTGAGCGAAAAGGAGAGACAGATCAAGAAACAGACAGCCCTGGTCGAACTGGTGAAGCATAAACCCAAGGCCACAAAAGAGCAGCTGAAGGCTGTCATGGACGATTTCGCAGCCTTTGTGGAAAAATGCTGTAAGGCAGACGATAAGGAGACTTGCTTTGCCGAGGAAGGAAAGAAACTGGTGGCTGCATCCCAGGCAGCTCTGGGACTGGGAGGAGGATCTGCCCCTACCTCAAGCTCCACTAAGAAAACCCAGCTGCAGCTGGAGCACCTGCTGCTGGACCTGCAGATGATTCTGAACGGGATCAACAATTACAAAAATCCAAAGCTGACCCGGATGCTGACATTCAAGTTTTATATGCCCAAGAAAGCCACAGAGCTGAAACACCTGCAGTGCCTGGAGGAAGAGCTGAAGCCTCTGGAAGAGGTGCTGAACCTGGCCCAGAGCAAGAATTTCCATCTGAGACCAAGGGATCTGATCTCCAACATTAATGTGATCGTCCTGGAACTGAAGGGATCTGAGACTACCTTTATGTGCGAATACGCTGACGAGACTGCAACCATTGTGGAGTTCCTGAACAGATGGATCACCTTCTGCCAGTCCATCATTTCTACTCTGACAGGCGGGGGGAGC 39 EETI-IIGC PRILMR CKQDSDCLAGCVCGPNGFCG from Knottin Database 40 AgRP fromGCVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCR- Knottin KLGTAMNPCSRT Database “-”indicates where mini protein can be formed 41 OmegaEDN--CIAEDYGKCTWGGTKCCRGRPCRC SMIGTN agatoxin CECTPRLIMEGLSFA fromKnottin Database “-” indicates where mini protein can be formed 42EETI-II GCXXXRGDXXXXXCKQDSDCLAGCVCGPNGFCG Library 43 EETI-IIGCXXXRGDXXXXXCSQDSDCLAGCVCGPNGFCG K15S Mutation Library 44 2.5F-GGTTGTCCAAGACCAAGAGGTGATAATCCACCATTGACTTGTTC (K15S)TCAAGATTCTGATTGTTTGGCTGGTTGTGTTTGTGGTCCAAATG mIgG2aFcGTTTTTGTGGTGGTCGACTAGAGCCCAGAGTGCCCATAACACA NucleicGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTC AcidCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAG SequenceATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAA 45 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPRVPITQNPCPP (K15S)LKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSED mIgG2aFcDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW AminoMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMT AcidKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSY SequenceFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK 46 2.5D-GGTTGTCCACAAGGCAGAGGTGATTGGGCTCCAACTTCTTGTTC (K15S)TCAAGATTCTGATTGTTTGGCTGGTTGTGTTTGTGGTCCAAATG mIgG2aFcGTTTTTGTGGTGGTCGACTAGAGCCCAGAGTGCCCATAACACA NucleicGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTC AcidCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAG SequenceATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCTGGGTAAA 47 2.5D-GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGEPRVPITQNPCP (K15S)PLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSE mIgG2aFcDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQD AminoWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEE AcidMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDG SequenceSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK 48 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGEPKSCDKTHTCP (K15S)PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV hIgG1FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG AminoKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ AcidVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS SequenceKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 49 2.5F-GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCGDKTHTCPPCPAP (K15S)ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY hIgG1FcVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC Fc UpperKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC HingeLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DeletionDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (ΔEPKSC) Amino Acid Sequence 502.5D- GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGEPKSCDKTHTC (K15S)PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV hIgG1FcKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG AminoKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQ AcidVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS SequenceKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 51 2.5D-GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGDKTHTCPPCPA (K15S)PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW hIgG1FcYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK Fc UpperCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT HingeCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DeletionDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (ΔEPKSC) Amino Acid Sequence 52hPD-1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDPWNPPTFFPALLVVT amino acidEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPG sequenceQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPR SAQPLRPEDGHCSWPL 53hPD-L-1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVE amino acidKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLK sequenceDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVKKCGIQDTNSK KQSDTHLEET 54hCTLA-4 MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPA amino acid VVLASSsequence RGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY LGIGNGTQIY VIDPEPCPDS DFLLWILAAVSSGLFFYSFLLTAVSLSKML KKRSPLTTGVYVKMPPTEPE CEKQFQPYFIPIN 55 hLAG3MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLP amino acidCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAP sequenceSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSL WLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRL EDVSQAQAGT YTCHIHLQEQ QLNATVTLAI ITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTEL SSPGAQRSGR APGALPAGHLLLFLILGVLS LLLLVTGAFG FHLWRRQWRPRRFSALEQGIHPPQAQSKIE ELEQEPEPEP EPEPEPEPEP EPEQL 56 hTIM3MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPG amino acidNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFR sequenceKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTR QRDFTAAFPR MLTTRGHGPA ETQTLGSLPD INLTQISTLANELRDSRLANDLRDSGATIRGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP 57 hB7-H3MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVG amino acidTDATLCC SFSPEPGFSLQLNLIWQLT DTKQLVHSFA sequence EGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFCFVSIRDFGSAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYQG YPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRN PVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLT GNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEEN AGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA 58 hB7-H4MASLGQILFWSIISIIIILAGAIALIIGFGISAFSMPEVNVDYNASSETL amino acidRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKV sequenceVSVLYN VTINNTYSCM IENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK 59 Anti-VEGFATGGGCTGGTCCCTGATCCTGCTGTTCCTGGTGGCTGTGGCCACTG clone B20- GCGTGCACTCTGAAGTGCAGCTGGTGGAATCTGGCGGCGGA 4.1.1 HeavyCTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCG ChainCCAGCGGCTTCAGCATCAGCGGCAGCTGGATCTTCTGGGTG nucleic acidCGCCAGGCCCCTGGAAAGGGCCTGGAATGGGTGGGAGCCA sequenceTCTGGCCTTTTGGCGGCTACACCCACTACGCCGACAGCGTGAAGGGCCGGTTTACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTCCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTATTGTGCCAGATGGGGCCACAGCACCTCCCCCTGGGCCATGGATTATTGGGGCCAGGGAACCCTCGTGACCGTGTCCTCTGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGCTCACTGTCCAGTGGTGTGCACACCTTCCCAGCTCTCCTCCAATCTGGCCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAACACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCA TCTCCCGGTCTCTGGGTAAA 60Anti-VEGF MGWSLILLFLVAVATGVHS EVQLVESGGGLVQPGGSLRLSCAASG clone B20-FSISGSWIFWVRQAPGKGLEWVGAIWPFGGYTHYADSVKGRF 4.1.1 HeavyTISADTSKNTAYLQMNSLRAEDTAVYYCARWGHSTSPWAMDY ChainWGQGTLVTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYF amino acidPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTI sequenceTCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERG SLFACSVVHEGLHNHLTTKTISRSLGK61 Anti-VEGF ATGGACATGAGAGTGCCCGCCCAGCTGCTGGGACTTCTGCTGCTGT clone B20-GGCTGCCAGGCGCCAGATGC GACATCCAGATGACCCAGAGCC 4.1.1 LightCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCAT ChainCACCTGTAGAGCCTCTCAGGGCGTGCGGACAAGCCTGGCC nucleic acidTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGAT sequenceCTACGATGCCAGCTCTCTGGCCAGCGGCGTGCCCAGCAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAAGAGCCCCCTGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT 62 Anti-VEGF MDMRVPAQLLGLLLLWLPGARCDIQMTQSPSSLSASVGDRVTITC clone B20-RASQGVRTSLAWYQQKPGKAPKLLIYDASSLASGVPSRFSGSG 4.1.1 LightSGTDFTLTISSLQPEDFATYYCQQSYKSPLTFGQGTKVEIKRAD ChainAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQ amino acidNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS sequence TSPIVKSFNRNEC 63Anti-CTLA- ATGGGCTGGTCCCTGATCCTGCTGTTCCTGGTGGCTGTGGCCACC 4 clone 9D9GGCGTGCACTCT GAAGCCAAGCTCCAGGAATCCGGCCCTGTG HeavyCTCGTGAAGCCTGGCGCCTCTGTGAAGATGAGCTGCAAGG ChainCCAGCGGCTACACCTTTACCGACTACTACATGAACTGGGTC nucleic acidAAGCAGAGCCACGGCAAGTCTCTGGAATGGATCGGCGTGA sequenceTCAACCCCTACAACGGCGACACCAGCTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAACTGAACAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGCGCCCGGTACTACGGCAGTTGGTTCGCCTATTGGGGCCAGGGCACCCTGATCACCGTGTCCACAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGCTCACTGTCCAGTGGTGTGCACACCTTCCCAGCTCTCCTCCAATCTGGCCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAACACCTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGAGCCCAGAGTGCCCATAACACAGAACCCCTGTCCTCCACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATCGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGCAGAAGAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGATCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTGGACCAGCAATGGGCGTACAGAGCAAAACTACAAGAACACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGGAAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAGGGTCTGCACAATCACCTTACGACTAAGACCATCTCCCGGTCTCT GGGTAAA 64 Anti-CTLA-MGWSLILLFLVAVATGVHS EAKLQESGPVLVKPGASVKMSCKAS 4 clone 9D9GYTFTDYYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKFKG HeavyKATLTVDKSSSTAYMELNSLTSEDSAVYYCARYYGSWFAYWG ChainQGTLITVSTAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEP amino acidVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITC sequenceNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLF ACSVVHEGLHNHLTTKTISRSLGK 65Anti-CTLA- ATGGACATGAGAGTGCCCGCCCAGCTGCTGGGACTTCTGCTGCTGT 4 clone 9D9GGCTGCCAGGCGCCAGATGC GACATCGTGATGACCCAGACCA Light ChainCCCTGAGCCTGCCTGTGTCCCTGGGAGATCAGGCCAGCATC nucleic acidAGCTGTCGGAGCAGCCAGAGCATCGTGCACAGCAACGGCA sequenceACACCTACCTGGAATGGTATCTCCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAAGGTGTCCAACCGGTTCAGCGGCGTGCCCGACAGATTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAGCCGAGGACCTGGGCGTGTACTACTGTTTTCAAGGCAGCCACGTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAAT GAGTGT 66 Anti-CTLA-MDMRVPAQLLGLLLLWLPGARC DIVMTQTTLSLPVSLGDQASISC 4 clone 9D9RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRF Light ChainSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTEGGGTKLE amino acidIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKID sequenceGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCE ATHKTSTSPIVKSFNRNEC

We claim:
 1. A method for treating cancer in a subject comprisingadministering to the subject an effective amount of an extendedpharmacokinetic (PK) interleukin (IL)-2 and an integrin-binding-Fcfusion protein, wherein the integrin-binding-Fc fusion protein comprises(i) an integrin-binding polypeptide comprising an integrin-binding loopand a knottin polypeptide scaffold; and (ii) an immunoglobulin Fcdomain, wherein the integrin-binding polypeptide is operably linked tothe Fc domain.
 2. The method of claim 1, wherein the extended-PK IL-2comprises a fusion protein.
 3. The method of claim 2, wherein the fusionprotein comprises an IL-2 moiety and a moiety selected from the groupconsisting of an immunoglobulin fragment, human serum albumin, and Fn3.4. The method of claim 3, wherein the fusion protein comprises an IL-2moiety operably linked to an immunoglobulin Fc domain.
 5. The method ofclaim 3, wherein the fusion protein comprises an IL-2 moiety operablylinked to human serum albumin.
 6. The method of claim 1, wherein theextended-PK IL-2 comprises an IL-2 moiety conjugated to a non-proteinpolymer.
 7. The method of claim 6, wherein the non-protein polymer is apolyethylene glycol.
 8. The method of claim 1, wherein theintegrin-binding polypeptide binds to a tumor-associated integrinselected from the group consisting of α_(v)β₃, α_(v)β₅, and α₅β₁, orcombination thereof.
 9. The method of claim 1, wherein theintegrin-binding polypeptide binds to α_(v)β₃, α_(v)β₅, and α₅β₁. 10.The method of claim 1, wherein the knottin polypeptide scaffoldcomprises at least three cysteine disulfide linkages or crosslinkedcysteine residues, and wherein the integrin-binding loop is adjacent tocysteine residues of the knottin polypeptide scaffold.
 11. The method ofclaim 10, wherein the integrin-binding loop comprises an RGD peptidesequence.
 12. The method of claim 10, wherein the knottin polypeptidescaffold is derived from a knottin protein selected from the groupconsisting of EETI-II, AgRP, and agatoxin.
 13. The method of claim 12,wherein the knottin protein is EETI-II.
 14. The method of claim 1,wherein the integrin-binding loop comprises an RGD peptide sequence andthe knottin polypeptide scaffold is derived from EETI-II.
 15. The methodof claim 1, wherein the knottin polypeptide scaffold is derived fromEETI-II and the integrin-binding loop comprises the sequenceX₁X₂X₃RGDX₇X₈X₉X₁₀X₁₁, wherein each X represents any amino acid, whereinthe loop is inserted between 2 cysteine residues in the EETI-II sequenceand replaces the native EETI-II sequence.
 16. The method of claim 15,wherein the integrin-binding loop is inserted after the first cysteinein the native EETI-II sequence.
 17. The method of claim 1, wherein theintegrin-binding polypeptide comprises the amino acid sequence set forthin SEQ ID NO: 42 or 43, wherein X₁ is selected from the group consistingof A, V, L, P, F, Y, S, H, D, and N; X₂ is selected from the groupconsisting of G, V, L, P, R, E, and Q; X₃ is selected from the groupconsisting of G, A, and P; X₇ is selected from the group consisting of Wand N; X₈ is selected from the group consisting of A, P, and S; X₉ isselected from the group consisting of P and R; X₁₀ is selected from thegroup consisting of A, V, L, P, S, T, and E; and X₁₁ is selected fromthe group consisting of G, A, W, S, T, K, and E.
 18. The method of claim1, wherein the integrin-binding polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 67-133. 19.The method of claim 1, wherein the integrin-binding polypeptidecomprises the amino acid sequence of SEQ ID NO: 94 or
 96. 20. The methodof claim 1, wherein the Fc domain is a human IgG1 Fc domain.
 21. Themethod of claim 1, wherein the integrin-binding polypeptide is operablylinked with or without a linker to the Fc domain.
 22. The method ofclaim 1, wherein the integrin-binding polypeptide is operably linked tothe N-terminus of the Fc domain.
 23. The method of claim 1, wherein theintegrin-binding polypeptide is operably linked to the C-terminus of theFc domain.
 24. The method of claim 1, wherein the integrin-binding-Fcfusion protein comprises the amino acid sequence of SEQ ID NO: 48, 49,50, or
 51. 25. The method of claim 1, wherein the extended-PK IL-2 andthe integrin-binding-Fc fusion protein are administered simultaneouslyor sequentially.
 26. The method of claim 1, further comprisingadministering an immune checkpoint blocker.
 27. The method of claim 26,wherein the immune checkpoint blocker is an antibody or antibodyfragment targeting a protein selected from the group consisting of PD-1,PD-L1, CTLA4, TIM3, LAG3, and a member of the B7 family.
 28. The methodof claim 27, wherein the immune checkpoint blocker is an antibody orantibody fragment targeting PD-1.
 29. The method of claim 27, whereinthe immune checkpoint blocker is an antibody or antibody fragmenttargeting CTLA4.
 30. The method of claim 1, wherein the cancer isselected from the group consisting of melanoma, leukemia, lung cancer,breast cancer, prostate cancer, ovarian cancer, colon cancer, renal cellcarcinoma, pancreatic cancer, cervical cancer, and brain cancer.
 31. Amethod for inhibiting growth and/or proliferation of tumor cells in asubject comprising administering to the subject an effective amount ofan extended-pharmacokinetic (PK) interleukin (IL)-2, and anintegrin-binding-Fc fusion protein, wherein the integrin-binding-Fcfusion protein comprises (i) an integrin-binding polypeptide comprisingan integrin-binding loop and a knottin polypeptide scaffold; and (ii) animmunoglobulin Fc domain, wherein the integrin-binding polypeptide isoperably linked to the Fc domain.